Simultaneous measurements of signal and background in inductively coupled plasma atomic emission spectrometry: effects on precision,limit of detection and limit of quantitation

Use of simultaneous measurements of the line intensity and the off-peak background intensity results in a significant improvement of repeatability at low concentrations, the limit of detection and the limit of quantitation in inductively coupled plasma atomic emission spectrometry. Simultaneous measurements have been performed using an echellebased dispersive system equipped with a custom segmented-array charge coupled device detector. Correlation between background signals, and shape of the curve of the RSD of the net signal as a function of the concentration is studied from both a theoretical and practical aspect. Concepts of limit of detection and limit of quantitation are discussed both in terms of RSD of the net signal and correlation. Results have been obtained using the Ba II 455 nm line in the visible region and the Ni II 231 nm line in the UV region. Improvements in the limit of detection and limit of quantitation are up to a factor of 10 and are in good agreement with theoretical values. The main limitation of the procedure is related to the accuracy of the background correction.     

抗酮洛芬多克隆抗体的制备及其间接竞争ELISA检测方法的建立

分别将酮洛芬与牛血清白蛋白(BSA)及卵清蛋白(OVA)偶联制得免疫原和包被原,经过免疫新西兰白兔制备多克隆抗体,抗体经纯化后效价为1:128000。使用自制的抗体,建立了测定酮洛芬的间接竞争酶联免疫吸附(ic-ELISA)新方法。ic-ELISA的线性范围为0.010～10.0μg/L,IC50为0.235μg/L,最低检测限为0.0040μg/L,线性回归方程为y=-22.97ρ+104.5(R2=0.980),与布洛芬、双氯酚酸的交叉反应率均小于4%,方法可用于水体中酮洛芬的检测。     

Validation study of a rapid ELISA for detection of histamine in tuna

Neogen Corp. has developed an improved Veratox histamine test kit for the detection of histamine in tuna tissue. The purpose of this study was to validate the method under the requirements of AOAC Research Institute (RI) Performance Tested Methods. Three AOAC Official Methods for histamine (954.04, 957.07, and 977.13) and one ELISA method have been performance tested by the AOAC RI. The most popular is AOAC Official Method 977.13, the fluorometric method, which is considered the reference method, but is complicated and time-consuming. The need for a rapid ELISA test kit to be validated by the AOAC RI exists.     

Validation of a high-performance liquid chromatographic method for the enantiospecific quantitation of zopiclone in plasma

Zopiclone is a hypnosedative with clinical effects similar to benzodiazepines but thought to have less potential for rebound insomnia and withdrawal effects. Zopiclone is administered as a racemic mixture, and an enantiospecific method of analysis of zopiclone in plasma is desirable in the study of pharmacokinetic drug interactions. We report a modification of an HPLC method reported by Foster et al. using a closely related structural analogue of zopiclone as internal standard. Zopiclone was detected at 306 nm and linear calibration curves were constructed in the range of 1.0-250 ng/mL for each enantiomer. The % CV at 2.5 ng/mL was 12.0% for (-)-zopiclone and 14.3% for (+)-zopiclone, and the limit of quantification of each enantiomer was 2.5 ng/mL. At higher concentrations, the coefficient of variation was less than 10%. The nominal concentration of quality control samples was predicted with an accuracy within a range of +/-11.6%. The method was used in the analysis of plasma obtained from psychiatric patients. One sample obtained following a non-fatal overdose with zopiclone contained the metabolites (-)-N-oxide zopiclone and both enantiomers of desmethyl zopiclone. The metabolite enantiomers were resolved on the column with retention times similar to zopiclone. The N-oxide metabolite co-eluted with internal standard.     

Validation of an HPLC method for quantitation of MDMA in tablets

An isocratic reversed-phase high-performance liquid chromatography (HPLC) method is developed and validated for the quantitation of 3,4-methylenedioxymethamphetamine (MDMA) in tablets. The chromatographic separation is achieved with potassium phosphate buffer (pH 3.2)-acetonitrile (9:1, v/v) as mobile phase, a Chromspher B column, and UV detection at 210 nm. The calibration curve is linear from 1.4 to 111 microg/mL. The percent relative standard deviation for intra- and interday precision studies is 2.7% each. The measurement uncertainty is estimated to 9%. The method is specific and successfully used for routine quantitation of MDMA in tablets.     

Potentiometric Cd-selective electrode with a detection limit in the low ppt range

A recently introduced model describing the response curves of polymer membrane ion-selective electrodes (ISEs) is used here to optimize a Cd²⁺-ISE with a view to achieve very low detection limits. In a first step, the selectivity behavior is determined for two membranes based on the ionophore, N,N,N′,N′-tetradodecyl-3,6-dioxaoctanedithioamide (ETH 5435), but having different concentrations of the ionophore and ion-exchanger. Based on these data, the optimal response curves are calculated with a model that takes into account the ion fluxes induced by interfering ions. The experimental results with different ionic backgrounds correspond very well with the predicted effects. The best lower detection limit of 10⁻¹⁰ M or 11 ppt Cd²⁺ is achieved at pH 7 with an ionic background of 10⁻⁴ M NaNO₃.     

High-resolution mass spectrometry method for the detection, characterization and quantitation of pharmaceuticals in water

The presence of pharmaceuticals in drinking water is an emerging environmental concern. In most environmental testing laboratories, LC-MS/MS assays based on selected reaction monitoring are used as part of a battery of tests used to assure water quality. Although LC-MS/MS continues to be the best tool for detecting pharmaceuticals in water, the combined use of hybrid high-resolution mass spectrometry (HRMS) and ultrahigh pressure liquid chromatography (UHPLC) is starting to become a practical tool to study emerging environmental contaminants. The hybrid LTQ-orbitrap mass spectrometer is suitable for integrated quantitative and qualitative bioanalysis because of the following reasons: (1) the ability to collect full-scan HRMS spectra with scan speeds suitable for UHPLC separations, (2) routine measurement of mass with less than 5 ppm mass accuracy, (3) high mass resolving power, and (4) ability to perform on-the-fly polarity switching in the linear ion trap (LTQ). In the present work, we provide data demonstrating the application of UHPLC-LTQ-orbitrap for the detection, characterization and quantification of pharmaceuticals and their metabolites in drinking water.     

Detection limit estimated from slope of calibration curve: an application to competitive ELISA.

This paper theoretically derives a general rule that while the slope of the semi-logarithmic plot (Y vs. log X) of a calibration curve varies depending on analyte concentration, X, the slope takes a specific value at the detection limit (L(D)). This rule holds good irrespective of the shape of the calibration curve (linear or non-linear) and in this paper, is applied to competitive ELISA (enzyme linked immunosorbent assay). The following relationship is deduced: slope of log-dose B/B0 at L(D) = [relative standard deviation (RSD) of blank responses] / 0.13. The L(D) obtained from the above-mentioned slope corresponds to the dose at which the RSD of dose estimates is 0.3 (= 30%). A commercial kit for 17alpha-hydroxyprogesterone is taken as an example.     

A study of the relation between the limit of detection and the limit of quantitation in inductively coupled plasma spectrochemistry

The limit of quantitation based on a repeatability threshold concept is compared with the limit of detection in induction coupled plasma atomic emission spectrometry (ICP-AES) and induction coupled plasma mass spectrometry (ICP-MS). A 5%-based limit of quantitation would normally correspond to 10 times the 3-σ based limit of detection. However, because of a possible lack of linearity of the calibration graph at low concentrations, some additional noise not taken into consideration and the possible use of time-correlated multichannel detection, this ratio of 10 cannot be used in every case. It is suggested that a suitable way of determining the limit of quantitation is to establish the plot of the percentage relative standard deviation (RSD) of the net signal as a function of the concentration in a range from the limit of detection to 50 times this limit.     

Direct competitive ELISA based on a monoclonal antibody for detection of aflatoxin B1. Stabilization of ELISA kit components and application to grain samples

A direct competitive enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody has been developed and optimized for detection of aflatoxin B₁ (AFB₁), and an ELISA kit has been designed. This immunoassay was highly specific, sensitive, rapid, simple, and suitable for aflatoxin monitoring. AFB₁ concentrations determinable by ELISA ranged from 0.1 to 10 μg L⁻¹. The IC₅₀ value was 0.62 μg L⁻¹. Recovery from spiked rice samples averaged between 94 and 113%. The effect of different reagents on the stability of HRP–AFB₁ conjugate solution was studied. The performance of a stabilized enzyme tracer in ELISA was determined and compared with that of a freshly prepared control solution of HRP–AFB₁ conjugate. The results showed that stabilizing media containing 0.02% BSA, 0.1% Kathon CG, and 0.05 mol L⁻¹ calcium chloride in 0.05 mol L⁻¹ Tris-HCl buffer (pH 7.2) maintained the activity of HRP–AFB₁ at a dilution of 1:1000 for a period of at least 12 months at room temperature whereas the reference conjugate solution without the additives lost its activity within a few days. Several additives were tested for their stabilizing effect on a monoclonal antibody (MAb) immobilized on the surface of polystyrene microtitre plates. It was shown that immobilized MAb, treated with post-coating solutions containing PVA, BSA, and combinations of these substances with trehalose, retained its activity for at least 4 months at 4°C, whereas the untreated MAb-coated plate lost its activity within 2 days.     

Validation of a novel LC-MS/MS method for the quantitation of colistin A and B in human plasma

A liquid chromatography/tandem mass spectrometry (LC-MS/MS) method, characterized by complete automation and high-throughput, was developed for the determination of colistin A and B in human plasma. All sample preparation procedures were performed by using 2.2 mL 96-deep-well plates, whereas robotic liquid-handling workstations were utilized for all liquid transfer steps, including solid-phase extraction (SPE). The whole preparation procedure was very rapid, whereas the method had a very short chromatographic run time of just 2 min. Sample analysis was performed by reversed phase LC-MS/MS, with positive electrospray ionization, using multiple reaction monitoring. The absence of available purified colistin A and B standards led to the development of a novel LC method with evaporative light-scattering detector for the determination of their stoichiometries in the standard mixture, along with its purity. The proposed bioanalytical method was fully validated and it was proven to be selective, accurate, precise, reproducible and suitable for the determination of colistin A and B in human plasma. It was applied successfully to a pharmacokinetic study for the determination of both analytes in samples of patients.     

测定茶叶中二氯联苯的酶联免疫吸附分析新方法

建立了测定茶叶中痕量3,4-二氯联苯(PCB12)的间接竞争酶联免疫吸附分析(ELISA)新方法。工作曲线方程为I=62.01+17.55lgρ,相关系数为0.9833,方法灵敏度IC50(抑制率50%)时PCB12的质量浓度达到0.207μg/L,检出限IC20(抑制率为20%时)PCB12的质量浓度为4.0 ng/L,交叉率小于1.0%。对市售铁观音茶叶做简单前处理后,用ELISA方法检测。加标回收实验显示回收率为94.5%~112.4%,相对标准偏差为4.0%~12%。     

Development of a combined technique using a rapid one-step immunochromatographic assay and indirect competitive ELISA for the rapid detection of baicalin

A colloidal gold conjugated anti-baicalin monoclonal antibody (anti-BA MAb) was prepared and used in an immunochromatographic assay (ICA) for BA in Scutellariae Radix and Kampo medicines. This competitive ICA uses an anti-BA MAb which shows a high specificity for BA and baicalein. Its advantages include a short assay time (15 min), no dependence on any instrumental systems, and it can detect BA in plant materials and Kampo medicines. The limit of detection for the ICA was found to be around 0.6 μg mL⁻¹of baicalin. Moreover, the usefulness of the combination of indirect competitive ELISA and the ICA using anti-BA MAb as a quality control method was confirmed for analysis of BA in Scutellariae Radix and Kampo medicines with a sufficient sensitivity (200 ng mL⁻¹ to 2 μg mL⁻¹), obtainable in an easy and timely manner.     

Validation of a simple liquid chromatographic method for determination and quantitation of residual ivermectin and doramectin in pig liver

A rapid and quantitative method for the extraction, derivatization, and liquid chromatography with fluorescence detection of ivermectin (IVM) and doramectin (DOM) residues in porcine liver was developed and validated. IVM and DOM were extracted from the liver samples with acetonitrile, the supernatant was evaporated to dryness at 37 degrees C under nitrogen, and the residue was reconstituted in 1-methylimidazole solution. After 2 min at room temperature, IVM and DOM were converted to a fluorescent derivative and then separated on a Hypersil ODS column. The derivatives of IVM and DOM were detected and quantitated with high specificity by fluorescence (excitation: 365 nm, emission: 475 nm). Abamectin was used as an internal standard. The mean extraction efficiencies from fortified samples (15 ng/g) were 75% for IVM and 70% for DOM. The limit of detection was 0.8 ng/g for both IVM and DOM.     

Comparison of monomeric and polymeric horseradish peroxidase as labels in competitive ELISA for small molecule detection

We have developed a simple and sensitive competitive enzyme-linked immunosorbent assay (ELISA) to determine aflatoxin B1 (as a model small analyte) and using streptavidin-polymeric horseradish peroxidase complex (SApolyHRP) as a label for signal amplification. The performance of the assay was evaluated by comparing it with the classical indirect competitive ELISA using HRP labeled anti-mouse IgG as the tracer antibody. The results indicate that the SApolyHRP-based competitive ELISA exhibits a typically 2.4-fold steeper slope of the linear working range of the calibration curve compared to the monomeric HRP based classical ELISA, i.e., the sensitivity was increased. The SApolyHRP conjugate causes a typically 19-fold stronger signal generation in comparison to the traditional HRP labeled anti-mouse IgG at the same concentration (25 ng mL^?1). Moreover, the SApolyHRP-based assay has a much wider linear range and a 3.8-fold better signal-to-noise ratio. Considering its simplicity, sensitivity and ease of operation, this competitive ELISA is considered to be a promising tool for small molecule immunodetection.

A routine method for the determination of plutonium in urine with a low detection limit

A radiochemical method for the determination of plutonium in urine is described. The steps involved are a) co-precipitation of plutonium, b) wet ashing, c) hydrolysis, d) extraction from 2M HNO₃ into capillary polypropylene columns coated with tri-n-octyl phosphineoxide 0.5M in toluene, and e) back-extraction of plutonium from the organic phase, f) electroplating onto stainless steel disks and spectrometry, since plutonium is extracted together with small amounts of uranium naturally occurring in urine. High quality deposits for spectrometry are obtained because iron interference is eliminated before back-extraction. The radiochemical recovery of²³⁹Pu is 55.6±7.5% and the detection limit is 1.0 mBq per liter of urine.     

Validation of a fast screening method for the detection of cocaine in hair by MALDI-MS

The sensitivity and specificity of a novel method of screening for cocaine in hair, based on matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry (MS), have been evaluated. The method entails a rapid extraction procedure consisting of shaking 2.5 mg pulverised hair at high frequency in the presence of an acidic solution (160 μL of water, 20 μL of acetonitrile and 20 μL of 1 M trifluoroacetic acid) and a stainless-steel bullet. Following centrifugation, the supernatant is dried under a nitrogen stream, and the residue is reconstituted in 10 μL of methanol/trifluoroacetic acid (7:3; v/v). One microlitre of the extract is deposed on a MALDI sample holder previously scrubbed with graphite; an α-cyano-4-hydroxycinnamic acid (matrix) solution is electrosprayed over the dried sample surface to achieve a uniform distribution of matrix crystals. The identification of cocaine is obtained by post-source decay experiments performed on its MH⁺ ion (m/z 304), with a limit of detection of 0.1 ng/mg of cocaine. A total of 304 hair samples were analysed in parallel by MALDI-MS and a reference gas chromatography-MS method. The obtained results demonstrate specificity and sensitivity of 100% for MALDI-MS. Evidence of cocaine presence was easily obtained even when hair samples exhibiting particularly low cocaine levels (<0.5 ng/mg) were analysed.     

A novel multiplex method for the simultaneous detection and relative quantitation of pollen allergens

Standardization of pollen protein extracts is essential in order to ensure efficiency and safety in allergy diagnosis and immunotherapy. In this paper, we have optimized a multiplex Western blotting method for the simultaneous detection of four olive pollen allergens (Ole e 1, Ole e 2, Ole e 5, and Ole e 9) on a single blot using a monoclonal antibody from mouse and three polyclonal antibodies raised in rabbit. We utilized unconjugated Fab antibody fragments for blocking rabbit primary antibodies, and fluorescence-based detection. These changes allowed an accurate and reliable comparative quantitation of these allergens among pollen-protein samples from six olive cultivars. In addition, we also tested the IgE-binding capacity of these pollen extracts by reprobing the same blot with a pool of sera from eight patients allergic to olive and detection with enzyme conjugated antibodies. A noticeable variability regarding allergen content and IgE-reactivity was found among the olive cultivars analyzed. Moreover, we could easily confirm the identity of some of the IgE-binding proteins by simply overlapping both fluorescence and chemiluminescence images. This method is versatile since it can be applied to other allergogenic plant species and extended to other allergens.     

Validation of PCR methods for quantitation of genetically modified plants in food.

For enforcement of the recently introduced labeling threshold for genetically modified organisms (GMOs) in food ingredients, quantitative detection methods such as quantitative competitive (QC-PCR) and real-time PCR are applied by official food control laboratories. The experiences of 3 European food control laboratories in validating such methods were compared to describe realistic performance characteristics of quantitative PCR detection methods. The limit of quantitation (LOQ) of GMO-specific, real-time PCR was experimentally determined to reach 30-50 target molecules, which is close to theoretical prediction. Starting PCR with 200 ng genomic plant DNA, the LOQ depends primarily on the genome size of the target plant and ranges from 0.02% for rice to 0.7% for wheat. The precision of quantitative PCR detection methods, expressed as relative standard deviation (RSD), varied from 10 to 30%. Using Bt176 corn containing test samples and applying Bt176 specific QC-PCR, mean values deviated from true values by -7to 18%, with an average of 2+/-10%. Ruggedness of real-time PCR detection methods was assessed in an interlaboratory study analyzing commercial, homogeneous food samples. Roundup Ready soybean DNA contents were determined in the range of 0.3 to 36%, relative to soybean DNA, with RSDs of about 25%. Taking the precision of quantitative PCR detection methods into account, suitable sample plans and sample sizes for GMO analysis are suggested. Because quantitative GMO detection methods measure GMO contents of samples in relation to reference material (calibrants), high priority must be given to international agreements and standardization on certified reference materials.     

Effect of a solvent on the parameters of the analytical signal,detection limit,and analytical range of the determination in analytical thermal lens spectrometry

The effect of the main parameters of the solvents (water, acetone, methanol, ethanol, acetonitrile, dimethyl sulfoxide, dichloromethane, toluene, and chloroform) that are most frequently used in the analytical practice on the characteristics of the thermal lens effect (increase in temperature because of optical heating and in the size of the region involved in heating) and on the detection limit and the lower determination limit of the thermal lens determination was considered, and the criteria of the selection of the medium for thermal lens experiments were discussed. It was demonstrated that the gain in these characteristics of the thermal lens determination in the given medium in comparison with the aqueous medium does not necessarily coincide with the strength of the thermooptical effect in this medium. It was demonstrated that the optimum conditions of measurements in the thermal lens detection are controlled not only by the absorption of the analyte and the reagents, but also by the intrinsic absorption of the solvent. Recommendations were given on the selection of the solvent for analytical thermal lens spectrometry.