Use Of External Standards Research Articles

Duplex digital droplet PCR for the determination of apricot kernels in marzipan

Marzipan is a mixture of almonds, sugar, and water. Almonds can be replaced by apricot kernels, which results in a similar product called persipan. Depending on the prices of almonds, profit can be maximized using apricot kernels instead of almonds. If not specified on the product, this kind of substitution is illegal and is prosecuted by official food control authorities. Likewise, also commercial buyers would like to know which product they bought. Real-time PCR systems for the determination of apricot DNA are already available, however, real-time PCR requires the use of external standards, which have a direct impact on the accuracy of the measurement. Currently, such standards are not available. In contrast to real-time PCR, digital PCR does not require external standards and exhibits a smaller measurement uncertainty as shown in recent publications. Therefore, we developed a duplex droplet digital PCR system to measure the proportion of apricot in marzipan without the use of external standards. We present validation data and results of an international proficiency test, underlining the applicability of this system.

Revisiting the Rius’ Standardless Method for the Quantitative X-Ray Diffraction Analysis of Mixtures of Inorganic Crystalline Phases

ABSTRACTAn alternative method for the standardless quantitative x-ray diffraction analysis of mixtures of inorganic crystalline phases proposed in the literature several years ago is presented. Our method requires only previously calculated μ*i values from tabulated data for all phases present in the mixtures. It does not require either the determination of calibration constants or the use of external standards, but it does require that the number of analyzed mixtures is larger than or equal to the number of phases present in them, and that the chemical composition of the mixtures are significantly different from each other. The integrated intensities of the chemically pure phases are estimated by a least-squares procedure from XRD data obtained from the mixtures. The method was tested against data published in the literature, with good results. Finally, a general expression for the “Normalized Height Law” proposed on an empirical basis by other researchers, has been theoretically derived.

A Systematic Investigation of Parameters Influencing Droplet Rain in the Listeria monocytogenes prfA Assay - Reduction of Ambiguous Results in ddPCR.

The droplet digital polymerase chain reaction (ddPCR) determines DNA amounts based upon the pattern of positive and negative droplets, according to Poisson distribution, without the use of external standards. However, division into positive and negative droplets is often not clear because a part of the droplets has intermediate fluorescence values, appearing as “rain” in the plot. Despite the droplet rain, absolute quantification with ddPCR is possible, as shown previously for the prfA assay in quantifying Listeria monocytogenes. Nevertheless, reducing the rain, and thus ambiguous results, promotes the accuracy and credibility of ddPCR. In this study, we extensively investigated chemical and physical parameters for optimizing the prfA assay for ddPCR. While differences in the concentration of all chemicals and the dye, quencher and supplier of the probe did not alter the droplet pattern, changes in the PCR cycling program, such as prolonged times and increased cycle numbers, improved the assay.

Coupling of size-exclusion chromatography with electrospray ionization charge-detection mass spectrometry for the characterization of synthetic polymers of ultra-high molar mass.

Coupling size-exclusion chromatography (SEC) with mass spectrometry (MS) allowed generation of a SEC calibration curve based on the analyte itself, which is more reliable than calibration based on non-related standards and faster than the use of the multiple detection mode. However, such SEC/MS couplings were limited to rather small synthetic polymers. Based on the concept of image current detection, charge-detection mass spectrometry (CDMS) coupled to electrospray ionization (ESI) is a useful method for weighing macro-ions from compounds with masses higher than one megadalton (MDa). Using columns designed to allow analysis of synthetic polymers of over 15 million Dalton in mass, performance of the SEC/ESI-CDMS coupling was evaluated for polyacrylamide (PAM, 5-6 MDa) and a poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (PAMPS, 2 MDa). The SEC/ESI-CDMS profiles were first compared with SEC-UV profiles: the systematic shift in retention time was assigned to the slightly different geometries of the two instrumental systems. The SEC/ESI-CDMS data were then compared with results obtained after the direct infusion of each sample into the ESI source. Both the shape of the molecular weight distribution and the mass values were similar with or without separation prior to ESI, and these values were consistent with data provided by the sample supplier. SEC/MS incorporating an online ESI-CDMS coupling was shown to be a rapid and efficient technique for the analysis of water-soluble synthetic polymers with ultra-high molecular mass in the megadalton range. The coupling also afforded an attractive solution for SEC calibration without the use of external standards.

Characterization of Anthocyanins in Perilla frutescens var. acuta Extract by Advanced UPLC-ESI-IT-TOF-MSn Method and Their Anticancer Bioactivity.

The anthocyanin extract from a domestic Perilla cultivar (Perilla frutescens var. acuta) were isolated and characterized with high mass accuracy and multi-dimensional fragmentation by means of ultra-performance liquid chromatography (UPLC) and electrospray ionization-ion trap-time of flight mass spectrometry analysis (ESI-IT-TOF-MSn). The new developed and applied LC-MS method focused on in-depth screening of anthocyanin compounds with similar structures which also provided a new approach of anthocyanin characterization without the use of external standards. Selective detection of interested anthocyanins was achieved utilizing extracted ion chromatogram (EIC) analysis, while MSn spectra were recorded to allow identification of the anthocyanin based on characteristic fragmentation patterns. Seven anthocyanins including one feruloyl (Cyanidin 3-O-feruloylglucoside-5-O-glucoside), two caffeoyl (Cyanidin 3-O-caffeoylglucoside-5-O-glucoside, Cyanidin 3-O-caffeoylglucoside-5-O-malonylglucoside) and four coumaroyl substituted anthocyanins (Cis-shisonin, Malonyl-cis-shisonin, Shisonin, and Malonyl-shisonin) were identified. Annexin-V FITC/PI flow cytometric assay was performed to analyze the influence of anthocyanin extract of P. frutescens var. acuta on cell apoptosis. The results suggested that Perilla anthocyanins can induce Hela cell apoptosis by a dose dependent manner.

Swift Quantification of <i>Fenofibrate</i> and <i>Tiemonium methylsulfate</i> Active Ingredients in Solid Drugs Using Particle Induced X-Ray Emission

The quantification of active ingredients (AI) in drugs is a crucial and important step in the drug quality control process. This is usually performed by using wet chemical techniques like LC-MS, UV spectrophotometry and other appropriate organic analytical methods. However, if the active ingredient contains specific heteroatoms (F, S, Cl…), elemental IBA like PIXE and PIGE techniques, using small tandem accelerator of 1-2 MV, can be explored for molecular quantification. IBA techniques permit the analysis of the sample under solid form, without any laborious sample preparations. In this work, we demonstrate the ability of the Thick Target PIXE technique for rapid and accurate quantification of both low and high concentrations of active ingredients in different commercial drugs. Fenofibrate, a chlorinated active ingredient, is present in high amounts in two different commercial drugs, its quantification was done using the relative approach to an external standard. On the other hand, Tiemonium methylsulfate which exists in relatively low amount in commercial drugs, its quantification was done using GUPIX simulation code (absolute quantification) The experimental aspects related to the quantification validity (use of external standards, absolute quantification, matrix effect,...) are presented and discussed.

Development of a robust, quantitative normal‐phase HPLC method for flavan‐3‐ols and procyanidin oligomers in chocolate and cocoa‐containing samples

A novel normal‐phase HPLC method for the quantitative determination of flavan‐3‐ols and procyanidins in chocolate and cocoa‐containing food products has been developed. Chromatographic separation based on degree of polymerization (DP = 1–10) was achieved on a diol stationary phase column (normal phase) with a binary gradient of acetonitrile (CH3CN:HOAc) and methanol‐water (CH3OH:H2O:HOAc). This novel methodology employs more environmentally friendly solvents than previously published methods and demonstrates greater resolution for larger oligomers. Quantitation was achieved with fluorescent detection (FLD: ex 230 nm/em 321 nm) and the use of external standards. With these solvents, fluorescence quantum yields were significantly greater than observed in previous methods. To have oligomers (DP1 −10) within the detection range of the instrument in a single run, and behave linearly over a wide range of concentrations, an FLD photomultipler tube gain gradient was employed. Sample preparation investigations were conducted to eliminate common chromatographic interferences and assess extraction efficiencies. Method performance was examined on selected cocoa‐containing foods (including NIST reference materials) with multiple analysts and laboratories. This method demonstrated accurate and reproducible quantitation of the flavanols and procyanidins in chocolate and cocoa‐containing products

International school accreditation

This article examines the nature of international school accreditation and seeks to place it within a wider accreditation framework encompassing both internal and external evaluation. The tensions between the self-study dimension of such accreditation and the use of external standards are explored, and the article concludes by indicating that the increasing use of generic indicators has the potential to lead to international schools that are less diverse and more homogenous.

Static headspace--gas chromatography: theory and practice

Static headspace--gas chromatography: theory and practice

Quantitative 1H NMR with External Standards: Use in Preparation of Calibration Solutions for Algal Toxins and Other Natural Products

We examine the use of external standards for quantitative measurement by 1H NMR of solution concentrations of natural products and other low molecular weight, hydrogen-containing compounds and show that precision and accuracy ca. 1% is obtainable with a commercial 11.7 T spectrometer when standards and analytes are contained in separate but identical sealed precision glass NMR tubes. Numerous factors contributing to the intensity of the NMR signals are evaluated. Precise measurements of 360 degrees pulse lengths for each sample provide direct corrections for variations in probe Q-factor that enable samples in different solvents to be compared, provided single-coil excitation and detection is used throughout. Samples need not be prepared in deuterated solvents if the 1H spectra of the solvents are simple enough for peak suppression by presaturation. The approach is particularly suitable for hazardous materials kept in sealed tubes and for the preparation of certified calibration solution reference materials for use with LC-MS and other techniques where deuterated solvents should be avoided.

Cell surface receptor‐antibody association constants and enumeration of receptor sites for monoclonal antibodies

Background Fluorescent markers (labeled antibodies) and flow cytometry are used to enumerate the average number of receptors (antigens) on formed bodies (cells) in whole blood by using a new method that avoids the extra steps of separating bound from unbound fluorescent markers or the use of external standards. Methods Mean channel fluorescence intensities of equilibrated marker-cell suspension mixtures, total concentrations of marker, and targeted cell counts obtained by standard cytometry procedures are used to complete the analyses for receptors per cell. Also, flow cytometric assays using competitive binding between fluorescent marker (CD4-RD1, CD8-FITC, CD3-FITC, CD3-RD1) and unlabeled antibody (CD4, CD8, CD3, CD3-dextran) for receptors on white blood cells in whole blood are described for determination of relative and specific binding constants of unlabeled/labeled antibody for targeted receptors. Results Ranges that were obtained for receptors per cell (lymphocytes) in normal blood donors were as follows: CD4, 4.9 × 104–1.5 × 105; CD8, 5.0 × 105–2.1 × 106; CD3, 6.6–7.8 × 105. Binding constants were highest for unlabeled CD4 antibody, 2.7 × 1010–2.1 × 1012 M-1, and then unlabeled CD3 antibody, 1.1 × 1010–1.9 × 1011 M-1. FITC- and RD1-labeled antibodies typically had binding constants that were 10-to 100-fold lower than the native antibodies. Conclusions Values of receptors per cell and binding constants obtained by the new method from flow cytometric analyses of mixtures of whole blood with FITC- or RD1-labeled CD4, CD8, and CD3 antibodies compare well with literature values determined by other methods. Cytometry 40:316–326, 2000 © 2000 Wiley-Liss, Inc.

Measurement of Procainamide and N-Acetylprocainamide in Serum by High-Performance Liquid Chromatography

We measured procainamide (I) and its metabolite, N-acetylprocainamide (II), in human serum samples by solvent extraction, high-performance liquid chromatography on a reverse phase column, and detection at 280 nm, with use of external standards. The method requires 0.2 ml of serum and is sensitive to 0.3 mg of I and 0.6 mg of II per liter of serum, with intra-assay standard deviations of 0.22 and 0.24 mg/liter, respectively, at 5 mg/liter (N=10) and inter-assay standard deviations of 0.63 and 0.81 mg/liter, respectively, at 7.5 mg/liter (CV 8.4 and 10.5%, respectively, n = 20). Concentrations measured by high-performance liquid chromatography and by an established fluorescence technique correlated well (r = 0.98 for I and 0.97 for II). No interfering substances were found in 20 randomly selected sera from patients receiving a large number of other drugs. Of the pure drug substances tested only sulfathiazole interfered with the assay of II. The method is therefore suitable for routinely monitoring these compounds in serum in a clinical laboratory. The high concentrations of the metabolite in a significant number of patients demonstrate the need to consider it as well as the parent drug as guides in optimizing dosage regiments for I.

Synchrotron X-ray fluorescence analysis of rock-forming minerals: 1. Comparison with other techniques; 2. White-beam energy-dispersive procedure for feldspars

Synchrotron X-ray fluorescence analysis (SXRFA) is compared with other microanalytical techniques including electron microprobe analysis (EMPA), particle-induced X-ray emission (PIXE), particle-induced gamma-ray emission (PIGE), nuclear reaction analysis (NRA) and secondary-ion mass spectrometry (SIMS). X-ray fluorescence spectra were taken of 21 feldspars and synthetic glasses to establish an experimental protocol and suitable standards for routine microprobe analysis of minor and trace elements. The white X-ray beam from a bending magnet of the 2.5-GeV National Synchrotron Light Source was apertured to 0.03 × 0.06 mm. The fluorescent X-rays were detected with an energy-dispersive system oriented at 90° to the incident beam. Spectra were obtained from each polished thick-section oriented at 45° and two sets of spectra were obtained for the crystalline feldspars at different orientations to evaluate the diffraction interference. Each spectrum was stripped using an interactive Gaussian-fitting program STRIP and elemental sensitivities were determined theoretically by the NRLXRF X-ray fluorescence analytical program without the use of external standards. Detection levels (part per million weight: 2σ) are: Ba and Ti (40), Mn, Fe, Cu, Zn, Ga, Ge, Rb, and Sr (2) and Pb (20). The higher values for Ba and Ti result in part from spectral overlap, and that for Pb from lower sensitivity for L spectra. The resulting standard-less elemental concentrations were compared with electron microprobe analyses of the same thick-section of each feldspar, and with atomic absorption spectrophotometry (AAS) of a fragment or slice. Concentrations determined by the three techniques agree to within ∼ ±20% with two exceptions. Rb contents determined by AAS agree with standard-less SXRFA and standard-controlled EMPA within only a factor of 2. Ba contents appear to be underestimated by standard-less SXRFA by ∼30% probably due to systematic errors in fundamental parameters used to obtain theoretical yields for Ba- L α fluorescence: hence, an external standard or an empirical correction is desirable. These results demonstrate that many trace elements in feldspars can be usefully determined by a simple standard-less technique referred to the major elements. Although the goal of this work has been the development of analytical protocols, valuable crystal-chemical data have been obtained including definitive evidence for substitution by Cu and Zn.

Determination of gas phase nicotine and 3‐ethenylpyridine, and particulate phase nicotine in environmental tobacco smoke with a collection bed – capillary gas chromatography system

AbstractA combined sampling and analysis technique for the determination of gas phase nicotine and 3‐ethenylpyridine, and of particulate phase nicotine in environmental tobacco smoke with capillary gas chromatography is reported. The major advantage of the technique is that all of the collected particulate phase material is analyzed by thermal desorption of the collected material rather than by analysis of only a fraction of the sample extracted from the collection medium. A Teflon filter microtube is used to collect particulate phase nicotine. This microtube is follwed by a small Tenax sorbent bed to collect gas phase nicotine and 3‐ethenylpyridine. After sampling, the Teflon filter is transferred to a clean glass tube and the tube becomes an insert for a modified packed column injector port where the material collected on the filter is heat desorbed to a cold capillary tubing trap. Gas phase nicotine and 3‐ethenylpyridine are also transferred from the Tenax to the GC column by thermal desorption from the Tenax sorbent bed. Gas phase nicotine and 3‐ethenylpyridine, and particulate phase nicotine are each determined by GC analysis of the desorbed material. Nicotine and 3‐ethenylpyridine are quantitated by the use of external standards. This technique is straightforward and can be used for semi‐real time determination of both gas and particulate phase compounds in environmental tobacco smoke. The results obtained by this technique compare well with those obtained by sampling with annular diffusion denuders.

Contribution to the statistical definition of the limits of reliable assay measurement

Different analytical limits for a calibration experiment can be calculated by the use of external standards. Various definitions for the limits of qualitative and quantitative analysis are compared, and an illustrative example is given.

Application of Tenax Trapping to Analysis of Gas Phase Organic Compounds in Ultra-Low Tar Cigarette Smoke

Abstract A method well suited to the analysis of gas phase organic compounds in ultra-low tar delivery cigarette smoke has been developed. The cigarette is smoked directly through a filter and a Tenax trap arranged in series. The components collected on Tenax are analyzed by thermal-desorption programmed-temperature gas chromatography. Components are then quantitated by the use of external standards. The smoke from high tar delivery cigarettes also can be analyzed by thermal desorption gas chromatography of portions of well mixed solid dilutions of the trapping Tenax. Deliveries of 34 gas phase components were determined for 4 ultra-low tar cigarettes having tar deliveries ranging from <0.01 to 3 mg tar/cigarette, and 24 components for 5 cigarettes delivering 7 to 45 mg tar/cigarette.

Ion molecule reactions at high pressure: recent proton affinities, gas phase acidities and hydrocarbon clustering results

After a brief historic survey of the development of high pressure ion molecule reaction measurements, research in three different areas is used to illustrate the possibilities of high pressure ion molecule equilibria studies. The proton transfer equilibria: B 1H + + B 2 = B 1 + B 2H + involving ∼30 bases B with proton affinities between water and ammonia were measured. A complete ladder of equilibria linking water to ammonia and often involving multiple cycles was obtained. Using the external standard PA(iso-butene) = 193 kcal/mole (Lossing, Semeluk, Tsang) one obtained absolute values for the proton affinities of the compounds. These include: water 169, hydrogen sulfide 171.9, CH 3OH 182.3, benzene 183.4, acetaldehyde 185.4, ethanol 186.8, dimethyl ether 190.1, acetone 194.6, diethyl ether 198.1 and ammonia 200.7 kcal/mole. Measurements of equilibria: A 1 − + A 2H = A 1H + A 2 − provide relative gas phase acidities of acids AH. Use of external standards leads to values for the bond dissociation energy-electron affinity difference, D(A-H) - EA(A). Such values were obtained for several carbon and nitrogen acids. The expected greater acidity of nitrogen acids RNH relative to carbon acids RNCH is found only in saturated compounds. Most strong nitrogen and carbon acids involve conjugated double bonds. For these systems the analogous carbon and nitrogen acids are of very similar acidity. It is suggested that this is due to resonance stabilization of the neutral (free) nitrogen acids. Some of the D(A-H) - EA(H) values obtained are: pyrolle 41.9, cyclopentadiene 39.1, acetanilide 35.3, phenyl acetone 36.2, acetylacetone 28, diacetamide 30.1, barbituric acid 16.3 kcal/mole. Studies of clustering reactions in hydrocarbon positive ions are interpreted in terms of three centre bonds. The systems whose energetics are considered are CH 5 + (CH 4) 2 and isomers of protonated ethane, propane and butane.

Factors affecting the use of external standards for spark source mass spectrometry

Factors affecting the use of external standards for spark source mass spectrometry