Similar Analytical Characteristics Research Articles

The comparison between light-scattering detectors based on LED and photodiode for immunoprecipitation assays of transferrin and ferritin

Undoubtedly, light-emitting diodes (LEDs) and photodiodes (PDs) are indispensable optoelectronic devices in modern analytical chemistry. LEDs can serve as either light emitters or detectors, thus being an alternative to the most popular detection systems consisted of PD. In this contribution, a comparison between LED-LED and LED-PD detectors, operating in turbidimetric and nephelometric modes, has been carried out for immunoprecipitation detection of transferrin and ferritin. The greatest emphasis was placed on the study of detectors responses under different measurement conditions including current powering an emitter, amplification gain in the case of PD as detector or the construction of detection cells designed for the Multicommutated Flow Analysis (MCFA). The assumption was to obtain the fully-mechanized system with simple but efficient detection system to enable the determination of iron-binding proteins occurring at different concentration ranges in human body. As a result, the optimized arrangements of LED-LED and LED-PD setups were characterized by similar analytical characteristics, enabling the determination of transferrin with the detection limit (LOD) of 0.2 mg/L and RSDs of 2.8–4.8% for LED-LED, and LOD of 0.1 mg/L and RSDs of 0.9–3.6% for LED-PD. In the case of ferritin detection, only the response of the LED-PD detector was statistically distinguishable in the range of 130–198 μg/L of protein with recorded analytical signal change of 20 mV value. The addition of polymer for signal enhancement provided the increase of response range to 107–253 μg/L, enabling the developed system for detection of pathological serum ferritin levels.

An analytical strategy to explore the multicomponent pharmacokinetics of herbal medicine independently of standards: Application in Gelsemium elegans extracts

The multicomponent pharmacokinetic study of herbal medicine is a great challenge due to the low plasma concentrations, large range of concentration scales, lack of authentic standards and uncertain interactions of the components. The aim of this work was to explore the in vivo pharmacokinetics of herbal medicine independently of authentic standards using an integrated analytical strategy. First, ion pairs of multiple components were tuned and selected, and then major parameters were optimized for derivative multiple reaction monitoring (DeMRM) by LC–MS/MS, which was combined with characterization of the chemical profiles of the herbal medicine by LC-QqTOF/MS. Second, different concentrations of herbal extracts were employed instead of authentic standards to construct calibration curves for the semiquantitative determination of multiple components in plasma. Taking Gelsemium elegans as an example, in addition to the fully validated and sufficient methodological results, a total of 27 alkaloid components, major bioactive constituents of Gelsemium elegans, were simultaneously monitored in pig plasma. The concentration-time profiles and pharmacokinetic properties of these 27 components were characterized. The absolute quantification of three components was compared with the results obtained using authentic standards, and the method showed very similar analytical characteristics, such as linearity, precision, accuracy, and the values of the pharmacokinetic parameters Tmax, Vd, Cl and MRT. This analytical strategy was found to be capable of assessing herbal pharmacokinetics independently of specific authentic compounds for each component. This study was the first attempt to systematically reveal the in vivo pharmacokinetics of Gelsemium elegans. This strategy and methodology will find widespread use in the quantitative pharmacokinetic analysis of multiple components independently of standards for herbal medicine, among other applications.

Quantification of desmosine and isodesmosine using MALDI-ion trap tandem mass spectrometry.

Desmosine (Des) and isodesmosine (Isodes), cross-linking amino acids in the biomolecule elastin, may be used as biomarkers for various pathological conditions associated with elastin degradation. The current study presents a novel approach to quantify Des and Isodes using matrix-assisted laser desorption ionization (MALDI)-tandem mass spectrometry (MS2) in a linear ion trap coupled to a vacuum MALDI source. MALDI-MS2 analyses of Des and Isodes are performed using stable-isotope-labeled desmosine d4 (labeled-Des) as an internal standard in different biological fluids, such as urine and serum. The method demonstrated linearity over two orders of magnitude with a detection limit of 0.02ng/μL in both urine and serum without enrichment prior to mass spectrometry, and relative standard deviation of < 5%. The method is used to evaluate the time-dependent degradation of Des upon UV irradiation (254nm) and found to be consistent with quantification by 1H NMR. This is the first characterized MALDI-MS2 method for quantification of Des and Isodes and illustrates the potential of MALDI-ion trap MS2 for effective quantification of biomolecules. The reported method represents improvement over current liquid chromatography-based methods with respect to analysis time and solvent consumption, while maintaining similar analytical characteristics. Graphical abstract ᅟ.

Comparison of copper labeling followed by liquid chromatography-inductively coupled plasma mass spectrometry and immunochemical assays for serum hepcidin-25 determination

Hepcidin-25 has been defined as the key biomarker in iron metabolism. This peptide binds to the iron transporter ferroportin to cause its degradation. Therefore, the need for specific, accurate and precise methods for the quantification of hepcidin-25 in biological fluids is dramatically increasing. In this regard, the use of rapid immunochemical methods that provide low limit of quantification is desired for routine clinical use. However, such fast methodologies should be first analytically evaluated and compared with alternative strategies to check for their advantages and limitations. Here we compare the use of a commercial immunochemical assay for hepcidin determination with a novel analytical approach based on Cu-labeling of the peptide followed by Cu determination using liquid chromatography (HPLC) and plasma mass spectrometry (ICP-MS). The figures of merit of both systems reveal similar analytical characteristics and both seem to be adequate for the determination of the peptide at biologically relevant concentrations in human serum samples. The analysis of a larger number of samples (n=50) by both techniques showed a good agreement in the concentrations found. Such finding permits to address the hepcidin recovery in the sample preparation procedure necessary for the HPLC-ICP-MS analysis in human serum that turn out to be 76–85%. Additionally, limitations due to cross-reactivity issues of the ELISA method could be addressed in some of the samples by using LC-ICP-MS and were confirmed by LC-Electrospray-MS.

Multiresidue analysis of organic pollutants by in-tube solid phase microextraction coupled to ultra-high performance liquid chromatography–electrospray-tandem mass spectrometry

In this work, in-tube solid phase microextraction (IT-SPME) coupling with ultra-high-pressure liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) multiresidue analytical method has been proposed for the first time for on-line enrichment of 9 analytes included in Water Frame Directive 2000/60/EC (WFD). The device was equipped with a GC TRB-5 capillary column, used as pre-concentration loop, and two conventional six-port injection valves. Water sample and desorption solvent volumes were tested. The optimum conditions were 4mL of processed sample followed by elution with 40μL of methanol. The analytes were detected with a mass spectrometer after being ionized positively using an electrospray ionization (ESI) source. The method presents good linearity over the range assayed, 0.025–2.5μg/L for chlorpyriphos and 0.25–25μg/L for the other tested compounds and LODs between 0.025μg/L and 2.5μg/L. Enrichment factors ranged from 2.5 to 10. Intra and inter-day variation coefficients were <26 and 31.6% respectively. Once validated, the method was applied to several water samples from different sources demonstrating that it achieves the on-line enrichment of the analytes with the advantage of minimum sample manipulation, and the identification and quantification of some organic pollutants in water samples in the range of low parts-per-billion. The method provided similar analytical characteristics as those obtained in the established couple IT-SPME–Capillary Liquid Chromatography (CapLC).

Analytical procedure for the determination of Ethyl Lauroyl Arginate (LAE) to assess the kinetics and specific migration from a new antimicrobial active food packaging

Ethyl Lauroyl Arginate (LAE) is a cationic tensoactive compound, soluble in water, with a wide activity spectrum against moulds and bacteria. LAE has been incorporated as antimicrobial agent into packaging materials for food contact and these materials require to comply with the specific migration criteria. In this paper, one analytical procedure has been developed and optimized for the analysis of LAE in food simulants after the migrations tests. It consists of the formation of an ionic pair between LAE and the inorganic complex Co(SCN)42− in aqueous solution, followed by a liquid–liquid extraction in a suitable organic solvent and further UV–Vis absorbance measurement. In order to evaluate possible interferences, the ionic pair has been also analyzed by high performance liquid chromatography with UV–Vis detection. Both procedures provided similar analytical characteristics, with linear ranges from 1.10 to 25.00mgkg−1, linearity higher than 0.9886, limits of detection and quantification of 0.33 and 1.10mgkg−1, respectively, accuracy better than 1% as relative error and precision better than 3.6% expressed as RSD. Optimization of analytical techniques, thermal and chemical stability of LAE, as well as migration kinetics of LAE from experimental active packaging are reported and discussed.

Gas chromatography – Optical fiber detector for assessment of fatty acids in urban soils

Fatty acids have been used as biomarkers of the microbial community composition of soils and they are usually separated and quantified by gas-chromatography coupled to a flame ionization detector (GC-FID). The aim of this study was to develop, validate and apply a methodology based on gas chromatography coupled to optical fiber detection (GC-OF) for screening five fatty acids used as indicators of fungal and bacterial communities in urban soils. The performance of the GC-OF methodology (optical fiber detector at 1550nm) was evaluated by comparison with the GC-FID methodology and it was found that they were comparable in terms of linear range, detection limit and analytical errors. Besides these similar analytical characteristics, the GC-OF is much cheaper than the GC-FID methodology. Different concentrations were determined for each fatty acid indicator which in turn varied significantly between the soil samples analyzed from Lisbon ornamental gardens. Additionally, the GC-OF showed a great potential as alternative for determination of eleven or more fatty acids in urban soils.

Simultaneous determination of penicillin G salts by infrared spectroscopy: Evaluation of combining orthogonal signal correction with radial basis function-partial least squares regression

In this study, a new method for the simultaneous determination of penicillin G salts in pharmaceutical mixture via FT-IR spectroscopy combined with chemometrics was investigated. The mixture of penicillin G salts is a complex system due to similar analytical characteristics of components. Partial least squares (PLS) and radial basis function-partial least squares (RBF-PLS) were used to develop the linear and nonlinear relation between spectra and components, respectively. The orthogonal signal correction (OSC) preprocessing method was used to correct unexpected information, such as spectral overlapping and scattering effects. In order to compare the influence of OSC on PLS and RBF-PLS models, the optimal linear (PLS) and nonlinear (RBF-PLS) models based on conventional and OSC preprocessed spectra were established and compared. The obtained results demonstrated that OSC clearly enhanced the performance of both RBF-PLS and PLS calibration models. Also in the case of some nonlinear relation between spectra and component, OSC-RBF-PLS gave satisfactory results than OSC-PLS model which indicated that the OSC was helpful to remove extrinsic deviations from linearity without elimination of nonlinear information related to component. The chemometric models were tested on an external dataset and finally applied to the analysis commercialized injection product of penicillin G salts.

Ultrasound-assisted extraction and derivatization of sterols and fatty alcohols from olive leaves and drupes prior to determination by gas chromatography–tandem mass spectrometry

A method for simultaneous determination of sterols and fatty alcohols in olive leaves and drupes based on ultrasound-assisted extraction and derivatization prior to individual identification–quantitation by chromatographic separation and mass spectrometry detection (single ion monitoring mode) is reported here. The sample preparation procedure involves the following steps: (i) leaching of the raw material accelerated by ultrasound; (ii) saponification of the leachate, also accelerated by ultrasound, and separation of the unsaponifiable matter; (iii) cleaning of the extract by solid-phase extraction; (iv) silylation of the target analytes—also assisted by ultrasound; (v) injection into the gas chromatograph for identification–simultaneous quantitation of the two families of compounds. Individual separation–determination of the fatty alcohols and sterols provide limits of detection (LOD) in the range 9.8 × 10 −2 to 2 μg/l and 5.0–6.0 μg/l, respectively. The LOQs range from 0.3 to 0.9 μg/l and 17.0 to 21.0 μg/l, and the linear dynamic ranges are between LOQ and 25.0 μg/ml. The between-day precision, expressed as relative standard deviation (RSD), ranges between 3.6 and 6.1% and the within-laboratory reproducibility, also expressed as RSD, between 6.4 and 9.2%. Within the study of the metabolomic profile of the unsaponifiable fraction in olive tree, the method has been applied to the determination of the target analytes in different varieties of olive trees cultivated in the same zone, so that differences in this unsaponifiable fraction can be attributed to characteristics of the target varieties. As compared with its European Union counterpart, the method is endowed with similar analytical characteristics and drastic shortening of the operational time.

Comparison between analytical performances of polyclonal and monoclonal electrochemiluminescence immunoassays for NT-proBNP

Background We evaluated the analytical characteristics of electrochemiluminescence (ECLIA) immunoassay for NT-proBNP (proBNP II, Roche Diagnostics, Germany) and compared its analytical performance to that of the previous polyclonal method. Methods We measured NT-proBNP in EDTA plasma samples of 177 consecutive cardiac patients (69 females and 108 males; mean age 62.2 ± 16.4 years, range 13 to 96 years) with monoclonal and polyclonal ECLIA methods following manufacturer's instructions using an Elecsys® 2010 analyzer. Results Monoclonal ECLIA method for NT-proBNP assay showed an imprecision (CV%) lower than 3% at the cut-off value (i.e., 150 ng/L). No significant interference was found in plasma samples containing high levels of hemoglobin, triglycerides or bilirubin. EDTA plasma showed slightly, but significantly lower NT-proBNP values than serum (on average − 6.3%) and lithium-heparinized plasma (on average − 3.9%) samples. Finally, a very close linear regression was found between the NT-proBNP values found by either monoclonal or polyclonal ECLIA method (monoclonal ECLIA = − 72.17 + 1.04 polyclonal ECLIA, n = 177, R = 0.993). Conclusions The monoclonal ECLIA method showed very similar analytical characteristics with slightly lower NT-proBNP results (on average − 2.5%) than the polyclonal ECLIA method. The difference between monoclonal and polyclonal methods seems to be too slight to change the reference range and decisional values for NT-proBNP assay, as previously assessed by the polyclonal ECLIA method.

Cross validation of multiple methods for measuring pyrethroid and pyrethrum insecticide metabolites in human urine

The objective of our study was to compare three vastly different analytical methods for measuring urinary metabolites of pyrethroid and pyrethrum insecticides to determine whether they could produce comparable data and to determine if similar analytical characteristics of the methods could be obtained by a secondary laboratory. This study was conducted as a part of a series of validation studies undertaken by the German Research Foundation's Committee on the Standardization of Analytical Methods for Occupational and Environmental Medicine. We compared methods using different sample preparation methods (liquid-liquid extraction and solid-phase extraction with and without chemical derivatization) and different analytical detection methods (gas chromatography-mass spectrometry (single quadrupole), gas chromatography-high resolution mass spectrometry (magnetic sector) in both electron impact ionization and negative chemical ionization modes, and high-performance liquid chromatography-tandem mass spectrometry (triple quadrupole) with electrospray ionization). Our cross validation proved that similar analytical characteristics could be obtained with any combination of sample preparation/analytical detection method and that all methods produced comparable analytical results on unknown urine samples.

Separation and Determination of Iron-Containing Proteins via Liquid Chromatography−Particle Beam/Hollow Cathode−Optical Emission Spectroscopy

Presented here is a method for the quantitative determination of iron-containing metalloproteins. Four iron-containing metalloproteins (transferrin, myoglobin, hemoglobin, and cytochrome c) were separated by high-performance liquid chromatography (HPLC) and determined through particle beam/hollow cathode-optical emission spectroscopy (PB/HC-OES) by the Fe (I) 371.9 nm optical emission. Parametric optimization of sample introduction, nebulization, and hollow cathode source conditions is performed for the suite of Fe-metalloproteins. Response curves for the Fe (I) emission were obtained under optimized conditions with detection limits for triplicate injections occurring on the nanogram level for iron ( approximately 24 ng) with variability of <7% RSD over the concentration range of 0.1-100 microg/mL iron in the metalloproteins. Response curves for S (I) emission yielded similar analytical characteristics. Optical emission detection of the liquid chromatography separations of the iron-containing metalloproteins demonstrates the feasibility of the PB/HC-OES system as a simple element-specific detector for liquid chromatography. The retention times of the four analytes are similar to those determined by UV absorbance (216 nm), demonstrating the ability of the PB interface to preserve the chromatographic integrity of the separation. Additionally, empirical formula calculations based on Fe (I) and S (I) emission response ratios provide a much higher level of specificity than single-element protein determination.

A comparative study of two sorbents for copper in a flow injection preconcentration system

Two solid sorbents based on the introduction of the reagent β-nitroso-α-naphthol in a polymeric matrix were employed as packing material in a flow injection preconcentration system for the determination of copper in food samples. The sorbents were prepared by two different means: functionalization of Amberlite XAD-4 by coupling β-nitroso-α-naphthol to a methylene spacer or impregnation of the copolymer with a solution containing the reagent. Both resins allowed copper preconcentration with very good analytical features. The enrichment factor obtained was 29 (both sorbents) for 60 s preconcentration time. For 180 s preconcentration time, enrichment factors of 58 (functionalized resin) and 61 (loaded resin) were obtained. Detection limits were 0.7 and 0.4 μg L −1 for functionalized and loaded resin, respectively. The procedure was applied to the analysis of bean, peanut and soybean samples. Studies performed in this work demonstrate that both functionalized and loaded NN-XAD have similar analytical characteristics and they are appropriate for employment in the on-line system.

An integrated bienzyme glucose oxidase–fructose dehydrogenase–tetrathiafulvalene-3-mercaptopropionic acid–gold electrode for the simultaneous determination of glucose and fructose

A bienzyme biosensor for the simultaneous determination of glucose and fructose was developed by coimmobilising glucose oxidase (GOD), fructose dehydrogenase (FDH), and the mediator, tetrathiafulvalene (TTF), by cross-linking with glutaraldehyde atop a 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM) on a gold disk electrode (AuE). The performance of this bienzyme electrode under batch and flow injection (FI) conditions, as well as an amperometric detection in high-performance liquid chromatography (HPLC), are reported. The order of enzyme immobilisation atop the MPA-SAM affected the biosensor amperometric response in terms of sensitivity, with the immobilisation order GOD, FDH, TTF being selected. Similar analytical characteristics to those obtained with single GOD or FDH SAM-based biosensors for glucose and fructose were achieved with the bienzyme electrode, indicating that no noticeable changes in the biosensor responses to the analytes occurred as a consequence of the coimmobilisation of both enzymes on the same MPA–AuE. The suitability of the bienzyme biosensor for the analysis of real samples under flow injection conditions was tested by determining glucose in two certified serum samples. The simultaneous determination of glucose and fructose in the same sample cannot be performed without a separation step because at the detection potential used (+0.10 V), both sugars show amperometric response. Consequently, HPLC with amperometric detection at the TTF–FDH–GOD–MPA–AuE was accomplished. Glucose and fructose were simultaneously determined in honey, cola softdrink, and commercial apple juice, and the results were compared with those obtained by using other reference methods.

Specific features of albumin interactions with hemiacetals of aflatoxins and sterigmatocystin

Aflatoxin B2a (AB2a), aflatoxin G2a (AG2a), and hemiacetal of sterigmatocystin have been shown to form immunoreactive conjugates with albumin. The conjugates were formed following incubation of solution mixtures at room temperature for 1 h, as demonstrated by spectrophotometry and enzyme immunoassay. Anti-AB2a antibodies reacted with AB2a, aflatoxin B1, and aflatoxin AB2 (100, 8.8, and 5.9%, respectively); a similar result was obtained for anti-AG2a antibodies reacting with AG2a, aflatoxin G1, and aflatoxin AG@2 (100, 2.5, and < 1.0%, respectively). Binding of anti-AB2a and anti-AG2a antibodies to solid-phase conjugates of AB2a or AG2a exhibited similar analytical characteristics.

Prussian Blue based screen printed biosensors with improved characteristics of long-term lifetime and pH stability

The promising advantages of Prussian Blue (PB) as catalyst and of the thick film screen printing technology have been combined to assemble sensors with improved characteristics for the amperometric determination of H 2O 2. PB-modified screen printed electrodes were applied to detect H 2O 2 at an applied potential of −0.05 V versus the internal screen printed Ag pseudoreference electrode, showing a detection limit of 10 −7 mol l −1, a linearity range from 10 −7 to 5×10 −5 mol l −1, a sensitivity of 234 μA mmol l −1 cm −2, and a high selectivity. Improved stability at alkaline pH values was also observed, which made possible their use with enzymes having an optimum basic pH. Then, the immobilisation of a single enzyme (glucose oxidase (GOD) or choline oxidase (ChOX)) or of two enzymes, acetylcholinesterase (AchE) coimmobilised with ChOX, has been performed on the surface of PB modified screen-printed electrodes (SPEs) using glutaraldehyde and Nafion®. ChOX has been selected as an example of enzyme working at alkaline pH. The choline biosensors showed a detection limit of 5×10 −7 mol l −1, a wide linearity range (5×10 −7–10 −4 mol l −1), a high selectivity and a remarkable long term stability of 9 months at 4 °C, and at least 4 weeks at room temperature. Similar analytical characteristics and stability were observed with the acetylcholine biosensors.

Multiresidue analysis of seventeen pesticides in wine by gas chromatography with mass-selective detection

We have developed a multiresidue method permitting the simultaneous quantitation of 17 pesticides in wine: dicloran, dimethoate, diazinon, chlorpyrifos-methyl, vinclozolin, carbaryl, methiocarb, dichlofluanid, parathion-ethyl, triadimefon, procymidone, myclobutanil, iprodione, imidan, dicofol, phosalone and azinphos-methyl. Solid-phase extraction of 0.5 ml of wine sample is followed by direct injection of 1 μl of the eluent onto a DB-5 MS gas chromatographic column followed by mass-selective detection using one target and two qualifier ions for each pesticide. The extraction and injection steps are carried out with automatic instrumentation. Good resolution of all compounds was achieved with a run-time approximating 23 min. Detection and quantitation limits were around 2 μg/l and 10 μg/l, respectively, with linear calibration curves up to 3 mg/l for most constituents. Recovery in half the compounds was >90%, and >80% in most of the remainder. Imprecision (relative standard deviation) was <10% for most pesticides and <18% in all. Further analytes can be added to the repertoire without difficulty. The method merits consideration together with four other multiresidue methods now available that offer similar analytical characteristics, slower run-times, and a different selection of analytes.

Development of monoclonal antibody-based immunoassays to the N-methylcarbamate pesticide carbofuran.

To produce monoclonal antibodies (MAbs) to the pesticide carbofuran, three compounds with carboxylic spacer arms of different lengths introduced at the carbamate group of the analyte structure were synthesized, conjugated to proteins, and used as immunizing haptens in mice. MAbs were subsequently characterized for affinity and specificity in the conjugate-coated format and in the antibody-coated format using newly synthesized compounds as heterologous assay haptens. Depending on the immunoreagent combination and assay format, competitive assays with I(50) values in the 1.2-10.2 nM (0.27-2.27 ng/mL) range were obtained. LIB-BFNB67 MAb in combination with the hapten BFNH, coupled either to horseradish peroxidase or to ovalbumin, was used to develop a direct and an indirect enzyme-linked immunosorbent assay, respectively. Optimized immunoassays displayed very similar analytical characteristics, with an I(50) value around 0.7 ng/mL and a limit of detection around 0.08 ng/mL. Both immunoassays were able to tolerate the presence of methanol up to a 15% concentration. Compounds very similar in structure to carbofuran (benfuracarb, furathiocarb, bendiocarb, and carbofuran-hydroxy) exhibited cross-reactivity values in the 18-37% range, but major N-methylcarbamate pesticides were not recognized by the MAb. These immunoassays should reasonably allow the rapid, low-cost, and sensitive determination of carbofuran in food, in soils, and in the environment at levels of regulatory and practical importance.

Quantitative analysis with d.c.‐ and r.f.‐glow discharge spectrometry

AbstractGlow discharge spectrometry (GDS) is a technique for rapid depth profiling and bulk analysis of materials. The latest instruments can be operated in either d.c. or r.f. modes. The d.c. mode provides analysis of conductive materials only, while the r.f. mode allows analysis of both conductive and non‐conductive samples. Until now, no theory for r.f.‐GDS has been available. The recently proposed d.c. theory, which assumes that emission yield depends only on pressure, is extended to r.f.‐GDS and is shown to be valid for the r.f. mode as well. A key to the success of the theory is that, in support of Duckworth and Marcus, the two modes are shown to have similar analytical characteristics, e.g. in comparing the two modes most elements examined had similar sensitivity factors and background equivalent concentrations. This will facilitate calibration of the light elements hydrogen, oxygen and nitrogen for use in the d.c. mode. Recommended procedures for bulk analysis, in either d.c. or r.f. modes, are presented and illustrated for manganese in steel. Both modes provide calibration curves of high linearity. Quantitative depth profiles of three commercial metallic‐coated steel samples are compared in d.c. and r.f. modes and the similarity of the quantitative profiles, given the very different lamp parameters operating between the modes, confirms that the theory works for both d.c. and r.f. The theory is then applied to data from a commercial prepainted metallic‐coated sample to provide the first quantitative GDS depth profile of a non‐conductive material.