Reproducibility Relative Standard Deviation Research Articles

Method Validation by Interlaboratory Studies of Improved Hydrophilic Oxygen Radical Absorbance Capacity Methods for the Determination of Antioxidant Capacities of Antioxidant Solutions and Food Extracts

Hydrophilic oxygen radical absorbance capacity (H-ORAC) is a method for evaluating antioxidant capacities of solutions of hydrophilic compounds. In this study, we improved the original method for H-ORAC determination, and evaluated the precision of the two improved methods (methods A and B) by interlaboratory studies using 5 antioxidant solutions and 5 food extracts as test samples. An interlaboratory study of method A, in accordance with the harmonized protocol, demonstrated satisfactory performance (intermediate precision relative standard deviations (RSD(int)) ranging from 4.6 to 18.8%; the reproducibility relative standard deviations (RSD(R)) ranging from 7.0 to 21.1%, and the HorRat values ranging from 0.40 to 1.93). However, methodological problems remained, and a further improved method, method B, was thus developed. An interlaboratory study of method B by 5 participating laboratories showed better intermediate precision and reproducibility (RSD(int) and RSD(R) ranging from 1.8 to 9.4%, and from 4.4 to 13.8%, respectively), and all HorRat values for the test samples were less than 1.3, suggesting good performance for the H-ORAC measurement.

Method for the Rapid Determination of Protein in Meats Using the CEM Sprint™ Protein Analyzer: Collaborative Study

A collaborative study was conducted to determine the protein content of raw and processed meat products by a protein-tagging and colorimetric technique. Meat products were prepared following AOAC Official Method 983.18 and analyzed using CEM Corporation's Sprint Rapid Protein Analyzer. Sprint provides protein results by combining an accurately weighed test portion with a known amount of dye-binding agent. The dye-binding agent binds with the lysine, histidine, and arginine, as well as the n-terminus of the proteins commonly found in raw meat and processed meat products. Results are displayed and reported by the Sprint as a percentage (g/100 g) of protein. Ten blind duplicate study samples were sent to 10 collaborating laboratories in the United States. The within-laboratory (repeatability) relative standard deviation (RSD(r)) ranged from 0.91 to 3.04%, and between-laboratories (reproducibility) relative standard deviation (RSDR) ranged from 1.50 to 3.41% for protein. The method is recommended for Official First Action.

Determination of Eight Major Bioactive Components in the Traditional Chinese Medicinal Preparation Xiaochengqi Decoction by High Performance Liquid Chromatography

High performance liquid chromatography was employed to determine the contents of eight marker bioactive components such as rhein, aloe-emodin, nobiletin, emodin, honokiol, magnolol, chrysophanol, and physcion in the traditional Chinese medicinal preparation, Xiaochengqi decoction. The separation was performed on a C18 column by stepwise gradient elution with water (1.1% v/v acetic acid)–acetonitrile (0.01min, 75:25; 15 min, 60:40; 30 min, 50:50; 40 min, 45:55; 55min, 30:70) as the mobile phase at a flow-rate of 1.0ml·min-1, with UV detection at 254nm. Eight regression equations showed good linear relationships between the peak area and the amount of each marker. The recoveries of the markers were 99.6, 99.5, 97.4, 97.7, 98.9, 97.9, 99.0 and 98.5%, respectively. The repeatability and reproducibility (relative standard deviation) of the method were less than 2.1% and 2.5%, respectively.

Liquid Chromatography Post-Column Oxidation (PCOX) Method for the Determination of Paralytic Shellfish Toxins in Mussels, Clams, Oysters, and Scallops: Collaborative Study

Sixteen laboratories participated in a collaborative study to evaluate method performance parameters of a liquid chromatographic method of analysis for paralytic shellfish toxins (PST) in blue mussels (Mytilus edulis), soft shell clams (Mya arenaria), sea scallops (Placopectin magellanicus), and American oysters (Crassostrea virginicus). The specific analogs tested included saxitoxin, neosaxitoxin, gonyautoxins-1 to -5, decarbamoyl-gonyautoxins-2 and -3, decarbamoyl-saxitoxin, and N-sulfocarbamoyl-gonyautoxin-2 and -3. This instrumental technique has been developed as a replacement for the current AOAC biological method (AOAC Official Method 959.08) and an alternative to the pre-column oxidation LC method (AOAC Official Method 2005.06). The method is based on reversed-phase liquid chromatography with post-column oxidation and fluorescence detection (excitation 330 nm and emission 390 nm). The shellfish samples used in the study were prepared from the edible tissues of clams, mussels, oysters, and scallops to contain concentrations of PST representative of low, medium, and high toxicities and with varying profiles of individual toxins. These concentrations are approximately equivalent to 1/2 maximum level (ML), ML, or 2xML established by regulatory authorities (0.40, 0.80, and 1.60 mg STX diHCl eq/kg, respectively). Recovery for the individual toxins ranged from 104 to 127%, and recovery of total toxin averaged 116%. Horwitz Ratio (HorRat) values for individual toxins in the materials included in the study were generally within the desired range of 0.3 to 2.0. For the estimation of total toxicity in the test materials, the reproducibility relative standard deviation ranged from 4.6 to 20%. A bridging study comparing the results from the study participants using the post-column oxidation (PCOX) method with the results obtained in the study director's laboratory on the same test materials using the accepted reference method, the mouse bioassay (MBA; AOAC Official Method 959.08), showed that the average ratio of results obtained from the two methods was 1.0. A good match of values was also achieved with a new certified reference material. The results from this study demonstrated that the PCOX method is a suitable method of analysis for PST in shellfish tissue and provides both an estimate of total toxicity, equivalent to that determined using the MBAAOAC Official Method 959.08, and a detailed profile of the individual toxin present in the sample.

Testing the suitability of different high-performance liquid chromatographic methods to determine aflatoxin M1 in a soft fresh Italian cheese

Aflatoxin M1 (AFM1) is a toxic undesirable compound in milk. AFM1 affinity for caseins causes a concentration effect during milk process for dairy transformation. In spite of this, no official method of analysis, nor maximum tolerance level for aflatoxin M1 in cheese have been established. Thus, the aim of this work was to test the suitability of different HPLC methods for the AFM1 quantification in soft cheese samples at three different contamination levels (low, medium and high, at respectively nearly 30, 100 and 250 ng/kg). Nine participants were selected among Italian laboratories accredited by the Italian accreditation body (ACCREDIA) for HPLC toxin analysis. They were asked to analyze samples applying the method routinely used. The different applied methods were compared, and precision and accuracy parameters were evaluated. The main differences among HPLC procedures were registered at the level of extraction step. The use of an enzymatic digestion for the extraction of the toxin from cheese seemed to be particularly advantageous and the use of immunoaffinity columns seemed to be determinant for the improvement of sensitivity at low contamination levels. In general, the applied methods well discriminated the 3 levels of contamination, even though they performed better at the medium and high concentration levels (100 and 250 ng/kg) than at the low one (30 ng/kg). In fact relative standard deviation for reproducibility at low level was higher (60.1%) than the same value at medium and high levels (22.8% and 28.9%, respectively).

Assessment of the genetic stability of GMOs with a detailed examination of MON810 using Scorpion probes

For the cultivation and maintenance of seeds, genetic mutations have to be avoided as much as possible. According to the Directive 2001/18/EC, genetic stability has to be proven for the approval of GMOs [1]. The investigation of genetic stability can be performed with various methods, but Southern blots are most commonly used. In this study, a different method, a real-time PCR involving Scorpion primer, was developed for the analysis of DNA rearrangements in GMOs. The advantage of this method is that already small changes in sequence can be detected. In addition, large numbers of samples can be tested at once in a single PCR run. Possible alterations in the region of the Scorpion primer can be detected by differences in the real-time PCR plots. The sequence that was analysed contained 130 nucleotides. The sensitivity of Scorpion primers to measure genetic stability was verified in experiments with plasmids that had only minor sequence alterations in the target region. Such small differences led to a total reduction of the fluorescence signal, even in heterozygous samples where the unaltered template was present as well. Scorpion primers were then used to investigate the transgenic sequences of 567 individual seeds of MON810 for maize at the 5′ and the 3′ region of the insert. In combination with SYBR green, the screening procedure allowed a selection of samples for further analysis. If a sample showed low fluorescence with the Scorpion primer but a normal amplification with SYBR green, then the integrity of the insert was checked by sequencing. The results, however, gave no indication of a mutation or rearrangement of MON810 seeds. In additional experiments, the reliability of the method was studied by determining several parameters that are required for a validation, e.g., specificity, cross-reactivity, dynamic range, correlation of the template concentration and the corresponding Ct values, PCR efficiency, relative repeatability standard deviation (RSDr) relative reproducibility standard deviation (RSDR).

Determination of metformin and other biguanides in forensic whole blood samples by hydrophilic interaction liquid chromatography–electrospray tandem mass spectrometry

Metformin is an anti-diabetic drug in the biguanide class which also includes phenformin and buformin. Because of the potential adverse effects of the biguanides, a reliable liquid chromatography-tandem mass spectrometry method using pneumatically assisted electrospray ionization was developed for the quantification of the drugs in both live and post-mortem human whole blood. The blood proteins were precipitated by the addition of a mixture of methanol and acetonitrile, and the extract was cleaned up by cation-exchange solid-phase extraction to eliminate ion suppression effects. The separation was performed by hydrophilic interaction liquid chromatography. Matrix-matched calibrants combined with isotope dilution of metformin were used for calibration. The detection limits were 0.01 mg/L for metformin and phenformin and the relative intra-laboratory reproducibility standard deviations were less than 6% at concentrations of 1-10 mg/L. The mean true recoveries were greater than 86%.

Determination of cathinones and related ephedrines in forensic whole-blood samples by liquid-chromatography–electrospray tandem mass spectrometry

A liquid-chromatography–tandem-mass-spectrometry method using pneumatically assisted electrospray ionisation (LC–ESI-MS/MS) was developed for the simultaneous determination of cathinone, methcathinone, ethcathinone, amfepramone, mephedrone, flephedrone, methedrone, methylone, butylone, cathine, norephedrine, ephedrine, pseudoephedrine, methylephedrine and methylpseudoephedrine in human live and post-mortem whole blood. The blood proteins were precipitated by the addition of methanol, and the extract was purified by ultrafiltration. The separation of diastereomeric ephedrines was achieved on an ethyl-linked phenyl column. Matrix-matched calibrants combined with the isotope dilution of selected substances were used for quantitative analysis. The relative intra-laboratory reproducibility standard deviations were generally better than 7% at concentrations of 20 μg/L, and the mean true recoveries were 87–106% in the concentration range of 10–250 μg/L. The detection limits were in the range of 0.5–3 μg/L. The cathinones were unstable in whole blood and sample extracts under neutral conditions, but the stability could be improved by the acidification of the sample matrix.

Simultaneous determination of five yellow dyes in foods by high performance liquid chromatography coupled with tandem mass spectrometry

A method based on high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) was developed for simultaneous determination of five yellow industrial dyes. The separations were performed with an Agilent ODS C18 column at a flow rate of 0.3 mL/min. The mobile phase was 5 mmol/L ammonium acetate (containing 0.1% formic acid)-acetonitrile (3:2, v/v). Under the optimized detection conditions, the linear ranges for Chrysoidine G and Basic Yellow 2 were 5.0-80.0 mg/L, and for Acid Orange I, Acid orange Il and Acid Yellow 36 were 10.0-160.0 microg/L. The limits of quantification for Chrysoidine G, Basic Yellow 2, Acid Orange I, Acid Orange II and Acid Yellow 36 were 20, 20, 40, 40 and 40 ng/g, respectively. The relative standard deviations of reproducibility of this method for retention time and peak area were no more than 0.50% and 2.14%, respectively. The method was applied to determine the recoveries of the above five dyes in chicken, bean products and yellow croaker were between 79.8%-95.2% with satisfactory results.

Inter-laboratory comparison of an analytical method for the determination of the feed additive semduramicin in poultry feed at authorised level by liquid chromatography single quadrupole mass spectrometry

A collaborative study was carried out according to internationally recognised guidelines in order to establish the performance characteristics of an LC/MS method for the determination of the feed additive semduramicin (SEM) in poultry feed at the level (20–25 mg kg−1) authorised within the European Union. Fifteen laboratories participated in the validation study, and all reported results. The content of SEM in the tested materials, provided as blind duplicates, ranged from 11.5 mg kg−1, which corresponds to half the mean authorised level, to 45.0 mg kg−1, which corresponds to twice the mean authorised level. All the materials were analysed by the participating laboratories using two different quantification approaches: standard addition and external standard calibration. The relative standard deviation of reproducibility (RSDR) for both quantification approaches varied from 8% to 18%, corresponding to HORRAT values ranging from 0.8 to 1.5, which were therefore in all cases below the critical value of 2.0. Consequently, the proposed analytical method and both quantification approaches can be considered to be fully validated and transferable to the control laboratories and applied for the determination of SEM in poultry compound feed at authorised level within the frame of official control. Further steps in the administrative procedure aiming to adopt the method as part of an ISO/CEN standard are currently ongoing.

Precision of the Cone Calorimeter and ICAL Test Methods

Repeatability and reproducibility are typically determined for standard fire test methods on the basis of an interlaboratory test program (round-robin) to define their precision. Significant differences in precision were found in the past between the repeatability and reproducibility of small-scale and intermediate- and large-scale fire test methods that all utilize oxygen calorimetry to measure heat release rate. Repeatability of heat release rate related measurements with small-scale apparatus has been found to be significantly better than the repeatability of the intermediate and large-scale apparatuses. In this paper, the results of two round-robins are compared on the basis of relative repeatability standard deviations and relative reproducibility standard deviations calculated for individual materials for individual test parameters. The first one was a cone calorimeter round-robin and the second was an intermediate-scale calorimeter (ICAL) round-robin. The objective and subjective factors that might have contributed to the differences between the two test methods in both repeatability and reproducibility were analyzed. The most important factors that caused the differences in the ICAL round-robin were higher theoretical uncertainty of the ICAL, inadequate pre-round-robin calibrations, the small numbers of participating laboratories and samples tested, prevalence of fire-retardant-treated materials, and possible failure of some of the laboratories to fully comply with the standard. These factors, especially the fact that the testing procedures and apparatuses in the participating laboratories may not have been in complete accordance with the standards, indicate that the results of the round-robins did not reflect the real precision of the test methods. However, the terminology using the two components of precision, repeatability and reproducibility, is maintained in this paper except where a specific distinction is noted. Recommendations are made on how to improve the results of future fire test round-robins.

Intra- and inter-laboratory variability in Real Dynamic Respiration Index (RDRI) method used to evaluate the potential rate of microbial self heating of solid recovered fuel

Microbial activity acts as primer in the self combustion process of solid recovered fuels (SRF) during their storage or transport. Thus, EU gave mandate to the European Committee for Standardization (CEN) to develop biological methods, (i.e. respirometric method) able to assess the risk of potential self combustion of SRF. Real Dynamic Respiration Index (RDRI) was chosen as official method, and a validation procedure was requested, to assure the quality of the results, when the method is applied for official purpose, i.e. repeatability and reproducibility detection. Two SRF coming from full-scale plants were analyzed for RDRI by three laboratories in six replicates. Results indicated a good precision of the method proposed in agreement with other biological methods, i.e. relative standard deviations of repeatability ranged from 16.7% to 17.8%, and a relative standard deviations of reproducibility ranged from 17.5% to 23.9%.

Cereals, cereals-based products and animal feeding stuffs - determination of crude fat and total fat content by the Randall extraction method: a collaborative study

A method for determining crude fat (fat A) and total fat (fat B) in cereals, cereal products and animal feeding stuff using 11 cereal and feed samples has been collaboratively studied, involving 15 participants from nine countries. The samples analyzed covered a range from about 0.5% to 26% crude fat (fat A) and from 1% to 27% total fat (fat B). Relative repeatability standard deviations ranged from 0.6% to 8.5% for crude fat and from 0.6% to 4.4% for total fat. The relative reproducibility standard deviations ranged from 1.0% to 22.6% for crude fat and from 2.6% to 13.4% for total fat. Analyzed samples comprised parboiled rice, whole wheat kernels, rye flour, sorghum kernels, couscous (durum wheat), a multicorn flourmix, croutons, cornbread, cattle feed, chickenfeed and pig feed. On basis of the obtained results the studied method has been published as international standard. Moller J (2010). Cereals, cereals-based products and animal feeding stuffs – determination of crude fat and total fat content by the Randall extraction method: a collaborative study. Quality Assurance and Safety of Crops & Foods, 2, 197–202

Determination of acidic and neutral therapeutic drugs in human blood by liquid chromatography–electrospray tandem mass spectrometry

A liquid chromatography–tandem mass spectrometry method using pneumatically assisted electrospray ionisation (LC–ESI-MS/MS) was developed for the simultaneous determination of paracetamol, naproxen, ibuprofen, etodolac, diclofenac, salicylic acid, lamotrigine, carbamazepine, 10-OH-carbazepine, phenytoin, phenobarbital, barbital, cyclobarbital, pentobarbital, amobarbital and caffeine in human live and post-mortem whole blood. The blood proteins were precipitated in a mixture of methanol and acetonitrile, and the extract was purified by ultrafiltration. The separation was performed on an ether-linked phenyl column with polar endcapping, and both negative and positive ESI were applied. Matrix-matched standards were used for calibration. The relative intra-laboratory reproducibility standard deviations were, in general, better than 8% at concentrations in the therapeutic range. The mean true recoveries were in the range 92–101% for the live blood and 84–101% for the post-mortem blood.

Determination of Walnut Protein in Processed Foods by Enzyme-Linked Immunosorbent Assay: Interlaboratory Study

Because food allergens from tree nuts, including walnuts, are a frequent cause of adverse food reactions for allergic patients, the labeling of foods containing ingredients derived from tree nuts is required in numerous countries. According to Japanese regulations, the labeling of food products containing walnuts is recommended. To ensure proper labeling, a novel sandwich ELISA kit for the determination of walnut protein in processed foods (Walnut Protein [2S-Albumin] Kit; Morinaga Institute of Biological Science, Inc.; "walnut kit") has been developed. We prepared seven types of incurred samples (model processed foods: biscuits, bread, sponge cake, orange juice, jelly, chicken meatballs, and rice gruel) containing 10 microg walnut soluble protein/g of food for use in interlaboratory evaluations of the walnut kit. The walnut kit displayed sufficient reproducibility relative standard deviations (interlaboratory precision: 5.8-9.9% RSDR) and a high level of recovery (81-119%) for all the incurred samples. All the repeatability relative standard deviation (RSDr) values for the incurred samples that were examined were less than 6.0%. The results of this interlaboratory evaluation suggested that the walnut kit could be used as a precise and reliable tool for determination of walnut protein in processed foods.

Validation of an analytical method for the determination of glyceroltriheptanoate (GTH) in processed animal by-products: Results of a collaborative study

The performance characteristics of a method for the determination of the marker substance glycerol triheptanoate (GTH) in processed animal by-products (ABPs) based on gas chromatography (GC) coupled to mass spectrometry (MS) were determined via a collaborative study. Within the European Union, GTH needs to be added to the portion of processed ABPs that must not enter the feed and food chain (Categories 1 and 2) at a minimum concentration of 250 mg kg−1 related to the fat fraction of the test samples analysed. The test materials included in the validation study consisted of three meat and bone meal (MBM) and three fat samples that contained GTH at different concentrations ranging from 61 to 455 mg kg−1. The relative standard deviation of repeatability (RSDr) varied from 3.4 to 7.8% and the relative standard deviation of reproducibility (RSDR) varied from 9.0 to 16.5%, corresponding to HORRAT values that were, in all cases, equal or below the critical value of 2.0. The estimated trueness expressed in terms of average concentration compared to the target concentrations of GTH in all test materials varied from 95 to 107%, confirming acceptable values for the trueness of the method. Based on the acceptable values for precision and trueness, the method is fit for the intended purpose and can be used for official control purposes to determine GTH in processed animal by-products from Category 1 and Category 2.

Determination of epinephrine in urine using multi-walled carbon nanotube modified with cobalt phthalocyanine in a paraffin composite electrode

This paper describes the development, electrochemical characterization and utilization of a cobalt phthalocyanine (CoPc), modified multi-walled carbon nanotube (MWCNT), and paraffin composite electrode for the quantitative determination of epinephrine (EP) in human urine samples. The electrochemical profile of the proposed composite electrode was analyzed by differential pulse voltammetry (DPV) that showed a shift of the oxidation peak potential of EP at 175 mV to less positive value, compared with a paraffin/graphite composite electrode without CoPc. DPV experiments in PBS at pH 6.0 were performed to determine EP without any previous step of extraction, clean-up, and derivatization, in the range from 1.33 to 5.50 μmol L −1, with a detection limit of 15.6 nmol L −1 (2.86 μg L −1) of EP in electrolyte prepared with purified water. The lifetime of the proposed sensors was at least over 1000 determinations with 1.7 and 3.1 repeatability and reproducibility relative standard deviations, respectively. Human urine samples without any purification step were successfully analyzed under the standard addition method using paraffin/MWCNT/CoPc composite electrode.

Determination of ochratoxin A in liquorice products using HPLC-based analytical methods. Part II: harmonised method and method validation study

On the basis of the outcome of an European proficiency test series conducted on behalf of the CAOBISCO (Association of the Chocolate, Biscuit and Confectionery Industries of the EU) expert group on ochratoxin A, a new harmonised method was developed for the analysis of ochratoxin A in liquorice extracts. This method works without the use of halogenated solvents because, as the proficiency test showed, an aqueous extraction solution can be used instead of, for example, chloroform, whose use is restricted in the EU. The main objective of this method validation study was to check the performance of this harmonised method. To carry out the method validation study, a set of three different test samples (one liquorice powder and two liquorice pastes) and a liquorice powder sample with an indicated range of ochratoxin A (a so-called sunshine sample) was distributed to 21 laboratories in ten countries throughout Europe and to one laboratory in the USA. The study was evaluated according to internationally recognised guidelines. In terms of its repeatability and reproducibility for determining ochratoxin A in liquorice extracts with a relative standard deviation for repeatability (RSDr) of between 6.68 and 19.95 and a relative standard deviation for reproducibility (RSDR) of between 17.39 and 29.08 the performance of the harmonised method was found to be in the accepted range of the EU directive for the analysis of mycotoxins in several foodstuffs.

Determination of Lycopene in Dietary Supplements and Raw Materials by High-Performance Liquid Chromatography: Collaborative Study

A collaborative study was conducted to evaluate the interlaboratory performance of an LC method for lycopene in dietary supplements and the raw materials commonly used in their manufacture. Twelve laboratories from six countries agreed to participate in the study. Results from 10 laboratories were received and are reported. Five dietary supplements, including both tablets and a softgel capsule with a lycopene content ranging from 25 microg to 25 mg per unit, and three raw materials, including gelatin-based beadlets, vegetarian beadlets, and a suspension in oil ranging from 5 to 20% lycopene, were analyzed as blind duplicates. In addition to the commercial products, two positive controls and a negative control were included in the study. For the raw materials studied, the repeatability relative standard deviations (RSDr) ranged from 1.49 to 5.13% for total lycopene, and the reproducibility relative standard deviations (RSDR) ranged from 3.84 to 9.21% with HorRat values from 1.23 to 3.24. For finished products, the RSDr ranged from 1.31 to 4.62%, RSDR from 4.28 to 10.5%, and HorRat values from 0.79 to 2.07. Corresponding values for all-trans-lycopene were significantly higher. It is recommended that the method be considered for Official First Action for all-trans- and total lycopene in finished products and raw materials.

Determination of sialic acids in infant formula by liquid chromatography tandem mass spectrometry

A liquid chromatographic/tandem mass spectrometric method using pneumatically assisted electrospray ionization (LC-ESI-MS/MS) was developed for determination of N-acetylneuramic acid and N-glycolylneuramic acid in infant formula. Reconstituted samples were hydrolysed in dilute sulfuric acid and deproteinized with acetonitrile. The extract was analysed directly without further clean-up by hydrophilic interaction chromatography. The substances were detected in negative ion mode and matrix matched standards were used for calibration. The relative intra-laboratory reproducibility standard deviation was better than 6% for both substances. An R² of 0.985 was obtained by comparison with a classical colorimetric assay based on reaction with resorcinol. The developed method is expected to be applied for accurate routine analysis of infant formulas.