Detection limit estimator for multivariate calibration by an extension of the IUPAC recommendations for univariate methods

A methodology is proposed to estimate the limit of detection (LOD) of analytical methods when multivariate calibration is applied. It tries to follow the same premises as the IUPAC methodology for univariate calibration. The mathematical support is given and algorithms such as partial least squares (PLS) regression, PLS2 and principal component regression (PCR) are used. Only multivariate raw data are used; that is, no surrogate univariate signal is deduced. Non-linearities are allowed. Near infrared (NIR) data of 5 component pseudo-gasoline samples together with simulated fluorescence synchronous spectra of binary mixtures (first order data) are used for evaluation. Experimental verification is performed using different kinds of data, namely: binary mixtures of bentazone and fenamiphos (very overlapped spectra, second order data) obtained by sequential injection (SI), and kinetic data of the reaction between the Fenton's reagent (FR) and pesticides such as atrazine, bentazone and alachlor (individual or binary mixtures, second order data). Results are always compared with independent methods previously proposed in the literature, based in the use of surrogate univariate signals. In general, similar results are found and no statistically significant differences seem to be present, except in a few cases when complex chemical systems are involved.     

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 method for predicting the precision, limit of detection and range of quantitation in competitive ELISA.

The mathematical model for predicting the precision, limit of detection (LOD) and range of quantitation (ROQ) in a competitive enzyme-linked immunosorbent assay (ELISA) proposed by Hayashi et al. (Anal. Chem., 2004, 76, 1295) was validated. The model describes the relative standard deviation (RSD) of concentration estimates by the RSDs of pipetting volumes of analyte, enzyme-conjugated antigen, antibody and substrate solutions, and the standard deviation (SD) of inherent absorbances between the wells in an ELISA plate. For 6 kinds of direct competitive ELISA kits, the LOD and ROQ predicted by the model agreed well with those obtained by experiments with real samples. It was also confirmed that the model is applicable to the prediction of uncertainty that depends on the pipetting error of the viscous antiserum solution. The model was demonstrated to be useful for estimating the LOD and ROQ of competitive ELISA.     

Estimation of the standard uncertainty of a calibration curve: application to sulfur mass concentration determination in fuels

The construction of a calibration curve using least square linear regression is common in many analytical measurements, and it comprises an important uncertainty component of the whole analytical procedure uncertainty. In the present work, various methodologies are applied concerning the estimation of the standard uncertainty of a calibration curve used for the determination of sulfur mass concentration in fuels. The methodologies applied include the GUM uncertainty framework, the Kragten numerical method, the Monte Carlo method (MCM) as well as the approximate equation calculating the standard error of prediction. The standard uncertainty results obtained by all methodologies agree well (0.172?C0.175?ng???L^?1). Aspects of inappropriate use of the approximate equation of the standard error of prediction, which leads to overestimation or underestimation of calculated uncertainty, are discussed. Moreover, the importance of the correlation between calibration curve parameters (slope and intercept) within GUM, MCM and Kragten approaches is examined.     

Detection of endocrine-disrupting pesticides by enzyme-linked immunosorbent assay (ELISA): application to atrazine

An overview of biological and toxicological effects of relevant endocrine-disrupting compounds is given. Special attention is paid to the determination of atrazine, a relevant pesticide that is considered an endocrine disrupter, by ELISA.     

Development of an indirect competitive ELISA for the determination of papaverine

Papaverine (1-(3,4-dimethoxybenzyl)-6,7-dimethoxyisoquinoline, PAP) is a member of the benzylisoquinoline sub-group of the opium alkaloids. It has been widely used for treating diseases like pulmonary arterial embolism and renal or biliary colic. In this paper, a specific conjugate of mono-demethylated papaverine-O-carboxylmethyl ether (MDMPAP-O-CME) and bovine serum albumin (BSA) was synthesized and used as the complete antigen (PAP-BSA), with which we successfully obtained a high-titer anti-PAP polyclonal antibody (pAb) by immunization of rabbits. The anti-PAP pAb showed high affinity to papaverine with an affinity constant (K_aff) of 7.3 × 10⁷ L/mol. With this antibody, we established a sensitive immunochemical method for the determination of papaverine based on indirect competitive enzyme-linked immunosorbent assay (ELISA). The optimal concentrations of the coated antigen (PAP-OVA) and purified pAb used in the ELISA were 5 and 1.2 μg/mL, respectively. The cross reactivity of other benzylisoquinoline derived substances, including 1-(3,4-dihydroxybenzyl)-7-hydroxy-6-methoxy-isoquinoline (6-methoxy-papaveroline, MPAPO), morphine (MP) and codeine (CD) were all lower than 1%. The linear range of the calibration curve was 0.1-1000 ng/mL. Normal human serum samples were spiked with known amount of papaverine and measured by the ELISA. Recoveries were between 102% and 105%. Papaverine content in a commercial papaverine hydrochloride injection sample was also determined using the established ELISA. Compared with the results given by the control experiment of HPLC, the recoveries of ELISA to detect injection samples were 102-110%. The limits of detection for synthetic serum samples and injection samples of papaverine hydrochloride were 0.25 and 0.06 ng/mL, respectively.     

Determination of the optimum working range from a calibration curve for colorimetric analysis with an "autoanalyzer"

A study has been made of the types and distribution of errors in determining the optimum working range from a calibration curve for automated analysis. Three error equations are derived for the fitting of a linear, quadratic or cubic curve, involving a thorough statistical analysis of the data. The errors from a set of silica and alumina standards, requiring a quadratic and cubic fit respectively, compare favourably with replicate analyses of standard rocks done on a previous occasion.     

Reusable plasmonic aptasensors: using a single nanoparticle to establish a calibration curve and to detect analytes

We demonstrate plasmonic aptasensors that allow a single nanoparticle (NP) to generate a calibration curve and to detect analytes. The proposed reusable aptasensors have significant advantages over conventional single-NP based assays in terms of sensitivity and reproducibility.     

Development of an electrochemical ELISA for the screening of 17 beta-estradiol and application to bovine serum

A sensitive electrochemical enzyme-linked immunosorbent assay (ELISA) for the detection of 17 beta-estradiol (17 beta-E2) was developed. Optimisation of two ELISA competition assays, using monoclonal or polyclonal antibodies anti-17 beta-estradiol, coupled with the electrochemical detection was firstly performed. The activity of the label enzyme (horseradish peroxidase) was measured electrochemically using 3,3',5,5'-tetramethylbenzidine as substrate. The use of the polyclonal antibody resulted in a more sensitive assay and the detection limit of the assay was estimated to be 20 pg ml-1. The analytical performances of the method were compared to those obtained using a dissociation enhanced lanthanide fluorescence immunoassay (DELFIA). Although sample extraction is not usually required by DELFIA, both extracted and non extracted samples were assayed. The comparison between the two screening techniques revealed similar results for the extracted samples and showed a comparable precision (RSD%), ranging from 6.2 to 13.4 and from 6.7 to 14.3 for DELFIA and ELISA, respectively. The results obtained by these screening assays were confirmed by liquid chromatography atmospheric pressure chemical ionisation tandem mass spectrometry which is currently used to confirm illegal hormone administration for regulatory purposes. The electrochemical enzyme immunoassay appears suitable as a screening tool for routine analysis of bovine serum estradiol and can be extended to other anabolic hormones using appropriate antibodies.     

Multiparametric curve fitting--VIII: The reliability of dissociation constants estimated by analysis of absorbance-pH curves

The program SPOPT estimates stability constants beta(pqr) and molar absorptivities epsilon(pqr) of all light-absorbing species M(p)L(q)H(r) by analysis of the absorbance-concentration (or absorbance-pH) curve. The program DCMINUIT estimates dissociation constants and molar absorptivities of protonated species. Both programs have been tested and compared with DCLET and LETAGROP-SPEFO for analysis of the overlapping equilibria of a triprotic acid. Computer plots of the residual-square-sum function are used to test the conditioning of parameters. Two approaches are made to formulation of the mathematical model, and several optimization algorithms are tested to find a reliable minimization procedure. The accuracy of ill-conditioned parameters is shown to be dependent on the precision of the absorbance measurements. General rules for investigation of A -pH curves are recommended.     

The calculation of vapor-liquid coexistence curve of Morse fluid: application to iron

The vapor-liquid coexistence curve of Morse fluid was calculated within the integral equations approach. The critical point coordinates were estimated. The parameters of Morse potential, fitted for elastic constants in solid phase, were used here to apply the results of present calculations to the determination of iron binodal. The properties of copper and sodium were considered in an analogous way. The calculations of pair correlation functions and isobars at liquid phase have shown that only for sodium these potential parameters allow one to obtain agreement with the measurements data. For iron another parameters are necessary to get this agreement in liquid phase. However, they give rise to very low critical temperature and pressure with respect to the estimates of other authors. Consequently, one can suppose that Morse potential is possibly inapplicable to the calculation of high temperature properties of non-alkali metals in disordered phases.     

Resolution of strongly competitive product channels with optimal dynamic discrimination: application to flavins

Fundamental molecular selectivity limits are probed by exploiting laser-controlled quantum interferences for the creation of distinct spectral signatures in two flavin molecules, erstwhile nearly indistinguishable via steady-state methods. Optimal dynamic discrimination (ODD) uses optimally shaped laser fields to transiently amplify minute molecular variations that would otherwise go unnoticed with linear absorption and fluorescence techniques. ODD is experimentally demonstrated by combining an optimally shaped UV pump pulse with a time-delayed, fluorescence-depleting IR pulse for discrimination amongst riboflavin and flavin mononucleotide in aqueous solution, which are structurally and spectroscopically very similar. Closed-loop, adaptive pulse shaping discovers a set of UV pulses that induce disparate responses from the two flavins and allows for concomitant flavin discrimination of ～16σ. Additionally, attainment of ODD permits quantitative, analytical detection of the individual constituents in a flavin mixture. The successful implementation of ODD on quantum systems of such high complexity bodes well for the future development of the field and the use of ODD techniques in a variety of demanding practical applications.     

A step-wise approach to the determination of the lower limit of analysis of the calibration line

A method is presented of stipulating the extension of the calibration line in analytical chemistry. The method is based on a combination of chemometric data interpretation and the calculation of the minimum standard deviation (STDEV) of experiments. From the depiction of the STDEV of a straight line versus the residuals, the more favorable concentration range of calibration could be obtained by iteration using only a few steps. This condition links the lower limit of analysis (LLA) to an upper limit of analysis (ULA), and, thus, completes the statistically appropriate extension of the calibration line. In addition, a minimum STDEV of measurement was expressed in terms of the STDEV on the slope (s _a ) and on the intercept (s _b ), and the calculation was performed by using a modified version of the law of propagation of errors (LPE). The method was validated on experiments of gas chromatography (GC), liquid chromatography (LC), electrochemistry, flow-injection analysis (FIA), atomic emission spectrometry (AES), flame atomic absorption spectrometry (FAAS), and inductively coupled-plasma mass spectrometry (ICP-MS). A novel definition of the ULA was proposed as the concentration where the response started to deviate significantly from linearity, also taking into account the level of uncertainty. The proposed method facilitates the determination of the concentration range of calibration and LLA in one working operation. In order to promote reliability, it was proposed that the number of repetitions of analysis should be high, preferably above 100, in order to fulfill the conditions of the central-limit theorem. The text was submitted by the author in English.     

A calibration curve at 2000 meters (A.S.L.): alpine valleys as field laboratories for teaching environmental monitoring to undergraduate students

High-altitude alpine valleys may be considered as ideal field laboratories for the interdisciplinary teaching of Environmental Sciences to undergraduate students in a Laurea degree, since different typologies of sampling sites (rivers, lakes, glaciers) may be found within walking distance, and students are encouraged to develop cooperative learning activities. Scientific data have been collected by 1st year students at the University of Insubria in Como during a teaching program in Ventina Valley and Caronno Valley near Sondrio (Italy). Analytical and geochemical results will be presented and discussed on the basis of organic deposition and water-rock interactions.     

Setting up a decision rule from estimated uncertainty: emission limit value for PCDD and PCDF incineration plants in Wallonia, Belgium

Complex analytical procedures are often required to prove the non-compliance with a specific legislation. In the case of a small overlap of the limit, integration of the method uncertainty in the decision-making process is essential. The decision rule proposed in Wallonia, Belgium, for the non-compliance of waste incineration plants with the EU limit value for PCDD and PCDF emissions is presented. The method uncertainty was estimated annually over 6 years from duplicate measurements using two top-down approaches. Depending on the congener, the standard uncertainty varies from 30 to 85%, with a good correlation between calculations. The analytical contribution was estimated using a bottom-up evaluation. The impact of the sampling step was deduced from the whole estimation and represents more than 80% of the total uncertainty budget. No optimisation is foreseen at this time because of practical field constraints. Based on the average fraction of each congener, the uncertainty associated with the measurement result has been established and shows a high stability over the years. Using this value, a guard band has been calculated and will be proposed to the regulatory body. Presented at the Measurement Uncertainty Symposium, Berlin, Germany, April 2008.     

Semiclassical initial value series representation in the continuum limit: application to vibrational relaxation

A recently formulated continuum limit semiclassical initial value series representation (SCIVR) of the quantum dynamics of dissipative systems is applied to the study of vibrational relaxation of model harmonic and anharmonic oscillator systems. As is well known, the classical dynamics of dissipative systems may be described in terms of a generalized Langevin equation. The continuum limit SCIVR uses the Langevin trajectories as input, albeit with a quantum noise rather than a classical noise. Combining this development with the forward-backward form of the prefactor-free propagator leads to a tractable scheme for computing quantum thermal correlation functions. Here we present the first implementation of this continuum limit SCIVR series method to study two model problems of vibrational relaxation. Simulations of the dissipative harmonic oscillator system over a wide range of parameters demonstrate that at most only the first two terms in the SCIVR series are needed for convergence of the correlation function. The methodology is then applied to the vibrational relaxation of a dissipative Morse oscillator. Here, too, the SCIVR series converges rapidly as the first two terms are sufficient to provide the quantum mechanical relaxation with an estimated accuracy on the order of a few percent. The results in this case are compared with computations obtained using the classical Wigner approximation for the relaxation dynamics.     

Detection of hazelnut in foods using ELISA: challenges related to the detectability in processed foodstuffs

Hazelnuts are widely used nowadays, and can pose a serious threat to allergic consumers due to cross-contamination that may occur during processing. This might lead to the presence of hidden hazelnut in foods. Therefore, reliable tests are needed to detect hazelnut, especially in processed foods. A hazelnut-specific indirect competitive ELISA based on polyclonal chicken antibodies was developed. The polyclonal antibodies were raised against modified hazelnut proteins in order to improve the detectability of hazelnut proteins in processed foods. The assay showed a detection limit of 1.36 microg hazelnut protein/mL of 5 mM urea in phosphate-buffered saline buffer (pH 7.4). Limited cross-reactivity with walnut and pecan nut was observed; no cross-reactivity was observed with other food ingredients. Blank cookies spiked before analysis showed recoveries of 73-107%. However, cookies spiked before baking showed that the detectability was severely decreased. Addition of lactose to the cookies, which led to more severe modification through the Maillard reaction, led to an increase in the detectability. These results indicate that using antibodies developed toward allergens modified through food processing-simulating reactions is a better approach for detection.     

Effect of lipophylic salts on ise detection limit: application to calixarene-based highly efficient potassium-selective electrodes

A new interpretative model for interpolation of potentiometric data from liquid membrane ion selective electrodes has been formulated; the model allows one to achieve information about partitioning equilibria establishing at the membrane-analysed solution interface of hydrophilic cations present in the membrane as counterions of exchanger lipophylic anions. The fundamental importance of such equilibria is related to their contribution to the detection limit of the electrodes. The soundness of the proposed model was positively verified by employing it in the characterization of new K(+)-selective electrodes based on a calixarenic ionophore. Although the validity of interpolation based on our model does not significantly differ from that obtained with the simpler model providing the inclusion of a constant parameter in the logarithmic argument of Nicolski-Eisenman's equation, the information obtained resulted to be analytically suitable for optimising the membrane composition and, as a consequence, the performance of the electrodes.     

Measurement uncertainty evaluation for a non-negative measurand: an alternative to limit of detection

The interpretation and reporting the results of measurements on materials where the concentration of the analyte is close to or may even be zero has been the subject of much discussion with the use of such concepts as limit of detection (LOD) and limit of quantification (LOQ). While these concepts have taken into account the measurement uncertainty, they have not utilised the fact that the value of the measurand, i.e., the concentration, is constrained to be zero or greater. Taking this into account the distribution of values attributable to the measurand can be derived from the probability density function (PDF) that determines the distribution of the observed values. When this PDF is normal the distribution of the values attributable to the measurand is a truncated t distribution with a lower limit of re-normalised so that the total probability is one, where x _m is the mean of the n observed values and s their standard deviation. When x _m much greater than then the distribution reverts to the unmodified t distribution. The probability that the value of the measurand is above or below a limit can be calculated directly from this truncated t distribution and the interpretation of the result does not require the use of concepts such as LOD and LOQ. Also it deals with the problem of negative observations. This Report was written by Alex Williams (e-mail: aw@camberley.demon.co.uk) for the Statistical Subcommittee and approved by the Analytical Methods Committee.     

Moment combined partial least squares (MC-PLS) as an improved quantitative calibration method: application to the analyses of petroleum and petrochemical products

A new quantitative calibration algorithm, called "Moment Combined Partial Least Squares (MC-PLS)", which combines the moment of spectrum and conventional PLS was proposed. Its calibration performance was evaluated for the analyses of three import petroleum and petrochemical products: gasoline, naphtha and polyol samples. The selected properties for these products included the research octane number (RON) and Reid vapor pressure (RVP) for gasoline, the distillation temperature at 10% (D 10%) for naphtha and the hydroxyl (OH) number for polyol. The major concept presented here used the moment to find the closest spectrum of a sample in a given dataset, and generate the difference spectrum and the corresponding difference in the property. These difference spectra and property differences were then used for PLS calibration. The moment has been employed in spectroscopic fields as a simple and effective "spectral feature characteristic" using just a few scalar values (moments). MC-PLS showed improved prediction performance over PLS for each case. In MC-PLS, the difference spectra generated using the moments were used as explained; therefore, additional detail in spectral variations can be utilized for calibrations. Additionally, the difference in the property was employed as reference data, so that its variation range was smaller when compared with that of the original property. Consequently, the MC-PLS performance could be better since the feature-enhanced spectra were used to model a narrower range of property variations. In the case of the D 10% prediction for naphtha, a non-linear prediction pattern that occurred in conventional PLS was effectively corrected using the MC-PLS method.