Candidate Certified Reference Material Research Articles

Development of an SI-traceable N-terminal pro-B-type natriuretic peptide certified reference material using structure-based impurity-corrected isotope dilution mass spectrometry approaches.

N-Terminal pro-B-type natriuretic peptide (NT-proBNP) is a pivotal biomarker for the diagnosis and prognosis of heart failure (HF). However, no SI-traceable certified reference material (CRM) or reference measurement procedure (RMP) is available for this biomarker, and so clinical testing results obtained in different laboratories cannot be traced to a higher-order standard, leading to incomparable measurements. Protein hydrolysis and protein cleavage isotope dilution mass spectrometry (AAA-IDMS and PepA-IDMS) were used to develop a CRM. Structurally related impurities were identified by high-resolution mass spectrometry. The quantitative AAA-IDMS results were corrected according to the amino acid compositions of the impurities. Using PepA-IDMS, two peptides from the proteolyzed product were confirmed as signature peptides. To obtain traceable and accurate results, the signature peptides were quantified using impurity-corrected AAA-IDMS. The candidate NT-proBNP solution was denatured and enzymatically digested using the Glu-C endoproteinase. The released signature peptides were measured using an isotopic dilution approach. The homogeneity and stability of the candidate CRM were characterized, and their uncertainties were combined with the value assignment process. The developed CRM can be considered a unique SI-traceable NT-proBNP reference material and is expected to be used as a primary calibrator for matrix NT-proBNP CRM development.

Study of the Stability and Homogeneity of Powdered Egg Matrix as a Candidate for Certified Reference Material for the Determination of Centesimal Composition

The full process of fresh egg preservation presents limitations and is associated with high transportation costs, while processed powdered eggs offer numerous advantages, such as high stability against degradation, providing extended shelf life, easy production, transportation, and favorable storage conditions. Traditional methods for determining the proximate composition of eggs and derivatives lack the capability to eliminate the matrix effect and do not guarantee adequate precision of the results. Therefore, Certified Reference Material (CRM) are the most suitable and effective tool to achieve this purpose. However, in Brazil, CRMs for powdered egg matrix are currently unavailable, although it is not possible to eliminate matrix effects, these can be included in the measurement uncertainty of each CRM. This study aimed to develop a candidate CRM for the powdered egg matrix with the requirements for the competence of testing and calibration laboratories (17034:2016) for proximate composition analyses. The analysis of variance (ANOVA) results for stability and homogeneity indicated no significant variation among the data obtained in the tests and the storage time of the material. Consequently, it was concluded that the powdered egg matrix is stable under simulated transportation conditions (short-term stability) as well as storage conditions (long-term stability) and exhibits adequate homogeneity for a CRM candidate.

Development and Application of Certified Reference Materials of Light Crude Oil for Water Content.

The water content of the crude oil needs to be measured accurately. The analytical methods that are used, including distillation and Karl Fischer coulometric titration (KFCT), require verification using certified reference materials (CRMs) of crude oil for the water content. In the present study, five CRMs of light crude oil with large volumes and various certified water contents (1-10 mg/g) were developed using the gravimetric method. Each candidate CRM was prepared individually, including the addition of pure water to the dried blank crude oil and weighing the masses of the added water and the blank crude oil. The water content of the blank crude oil was measured using azeotropic distillation-KFCT (AD-KFCT). The certified water content for the CRM was calculated by using the water content of the blank crude oil, the masses of the added pure water, and the blank crude oil. The certified water content and the expanded uncertainty (U, k = 2) of the five CRMs were 1.12 (0.04), 2.14 (0.06), 3.14 (0.10), 5.14 (0.15), and 10.05 (0.27) mg/g. These CRMs were used to verify the accuracy of analytical methods, including distillation, KFCT, and AD-KFCT methods.

Development of a Pure Certified Reference Material of D-Mannitol.

A new certified reference material (CRM) of D-mannitol (GBW(E) 100681) has been developed in this study. We describe the preparation, structure determination, characterization, homogeneity study, stability study, as well as uncertainty estimation. The main component was 99.91% ± 0.01%. The moisture content of the candidate CRM was 0.036% ± 0.002%, as measured by Karl Fischer titration. The nonvolatile and volatile impurities in the candidate CRM were all much less than 0.01%, which was determined by the ICP-MS and headspace GC-FID methods, respectively. The purity of the D-mannitol CRM was 99.9% ± 1.1% (k = 2), as measured by the two independent approaches involving the mass balance method (MB) and quantitative nuclear magnetic resonance technique (qNMR). The D-mannitol CRM was stable during the monitoring period for each temperature. It is stable for up to 48 months at room temperature and 28 days at 50 °C. The uncertainty was evaluated by combining the contributions from characterization, homogeneity, and stability. The developed D-mannitol CRM would effectively support method validation and proficiency testing, as well as effectively guarantee the accuracy, reliability, and comparability of results.

Development of pure certified reference material of squalene

A new certified reference material (CRM) of squalene (GBW(E) 091179) has been developed using a mass balance (MB) approach in conjunction with quantitative nuclear magnetic resonance (qNMR) technique. In compliance with ISO guidelines, the candidate CRM has been investigated for preparation, structure determination, characterization, homogeneity study, stability studies, as well as uncertainty evaluation. The squalene CRM has a certified value of 99.4 %, whose expanded uncertainty is 0.2 % (k = 2). Under a −20 °C condition, the squalene CRM is stable for no less than 12 months, while at 50 °C, its stability duration is 14 days. The uncertainty was evaluated by combining the contributions from characterization, homogeneity, and stability. The developed squalene CRM may be an effective support for method validation, intercomparison in the laboratory, quality control, and metrological traceability used in the food, pharmaceutical, and cosmetic industries.

Preparation and evaluation of certified reference materials for crude protein, crude fat, and crude ash in feed

Three new certified reference materials (CRMs) for crude protein, crude fat, and crude ash in animal feed were developed and characterized according to ISO 17034:2016 and ISO Guide 35:2017. The Kjeldahl, Soxhlet extraction, and ashing methods were employed to quantify these three components in candidate CRMs for swine feed, poultry feed, and fish feed. To improve the precision, the measurement methods were optimized on the basis of Directive 93/28/EEC, Directive 98/64/EC, and Directive 71/250/EEC and further validated using two existing CRMs (BCR-708 and BCR-709). The homogeneity, short- and long-term stability, and moisture content were evaluated. The certified values and their expanded uncertainties (k = 2.00) were determined through nine laboratories. The results for crude protein, crude fat, and crude ash were 14.7 ± 0.4, 5.19 ± 0.24, and 6.23 ± 0.14% for swine feed CRM; 22.7 ± 0.7, 5.16 ± 0.39, and 5.74 ± 0.18% for poultry feed CRM; and 35.6 ± 0.9, 9.29 ± 0.40, and 8.70 ± 0.25% for fish feed CRM, respectively. These three CRMs have been used in the comparison of Chinese feed quality testing laboratory systems and proved suitable for evaluating quality control in laboratories.

Production of Bauxite Certified Reference Material (BARC‐B1201) for Nine Property Values (Al2O3, Fe2O3, SiO2, TiO2, V2O5,MnO, Cr2O3, MgO and LOI) Traceable to SI Units

The National Centre for Compositional Characterisation of Materials (NCCCM) / Bhabha Atomic Research Centre (BARC) and National Aluminium Company Limited (NALCO), India have produced an Indian origin bauxite certified reference material (CRM), referred to as BARC‐B1201, certified for major (Al2O3, Fe2O3, SiO2, TiO2, loss on ignition ‐ LOI) and trace contents (V2O5, MnO, Cr2O3, MgO). Characterisation was undertaken by strict adherence to ISO Guides. A method previously developed and validated in our laboratory, using single step bauxite dissolution and subsequent quantitation (of Al2O3, Fe2O3, SiO2, TiO2, V2O5, MnO, Cr2O3 and MgO) by ICP‐AES (SSBD ICP‐AES) was used for homogeneity studies and an inter‐laboratory comparison exercise (ILCE) of the candidate CRM. LOI was determined by thermo‐gravimetric analysis. Property values were assigned after an ILCE with participation from seventeen reputed government and private sector laboratories in India. The CRM was certified for nine property values: Al2O3, Fe2O3, SiO2, TiO2, V2O5, MnO, Cr2O3, MgO and LOI, which are traceable to SI units.

Production of a matrix certified reference material for measurement and risk monitoring of clenbuterol in mutton.

A novel matrix certified reference material (CRM) for clenbuterol in mutton (GBW 10216) was developed to assist measurement and risk monitoring of clenbuterol in mutton. The candidate CRM raw samples were obtained by oral administration of clenbuterol and investigating the pharmacokinetics of clenbuterol in sheep. A high-precision isotope dilution coupled with liquid chromatography tandem mass spectrometry (LC-ID-MS/MS) method was established and assigned the value of clenbuterol in mutton powder through combined detection of nine inter-laboratories. The certified value with expanded uncertainty was 21.1 ± 2.2μg/kg (k = 2, 95% confidence) for clenbuterol in mutton. The prepared matrix CRM was sufficiently homogeneous between and within bottles. The long-term stability of clenbuterol in mutton powder was evaluated for 12months at -20℃ and short-term stability for 7days at 4℃ and 50℃. The uncertainties originating from characterization, homogeneity, and stability were systematically analyzed and evaluated. The prepared matrix CRM can be applied for proficiency testing and nationwide risk monitoring programs to guarantee the accuracy and comparability of clenbuterol measurement results in mutton.

APMP-APLAC joint proficiency testing programs for elemental analysis in food with metrological reference values: Assessment of participants' performance considering measurement uncertainties

The Asia Pacific Metrology Program (APMP) and the Asia Pacific Laboratory Accreditation Cooperation (APLAC) joint Proficiency Testing (PT) programs for toxic elements such as cadmium (Cd) and lead (Pb) or nutritional elements such as iron (Fe) and zinc (Zn) in food were organized by the Korea Research Institute of Standards and Science (KRISS) with the aim of enhancing the quality of measurement and metrological traceability in various economies of the Asia Pacific region by evaluating the performance with rigorous evaluation. Three APMP-APLAC joint PT programs for elemental analyses were carried out by KRISS sequentially, where candidate certified reference materials (CRMs) were used as the PT materials and metrologically traceable certified reference values (RVs) were used as the PT assigned values for the evaluation of participants' results, which allows reliable evaluation of participant performance. This article describes the operation of the PTs and the overall performance of the participating laboratories. The effectiveness of these joint PT programs and trends in PT performance assessment are also discussed. These PT programs confirm the significant importance of using the metrologically traceable RVs instead of the consensus values from participants as the PT assigned value for reliable assessment. The lack of understanding of the concept of coverage factor, degree of freedom, standard uncertainty, and expanded uncertainty was revealed by some participants in these PT programs. Interpreting the zeta-scores or En scores, which are derived by using measurement uncertainties, in conjunction with the z-scores is highly meaningful for assessing participants' ability in measurement capabilities and measurement uncertainty evaluation. Assessment of participants' performance considering measurement uncertainties helps the participants to check how reasonable their measurement uncertainty estimation was. The results of PTs also demonstrated that these PT programs are useful for improving the measurement capability of the laboratories, whereas more capability-building in uncertainty evaluation is required for further improvement.

Development of Certified Reference Material for Amino Acids in Dried Blood Spots and Accuracy Assessment of Disc Sampling.

To achieve the measurement reliability of amino acids used as diagnostic markers in clinical fields, establishing a reference measurement system is required, in which certified reference materials (CRMs) are an essential step in the hierarchy of measurement traceability. This study describes the development of dried blood spot (DBS) CRMs for amino acid analysis with complete measurement traceability to the International System of Units (SI). Six essential amino acids—proline, valine, isoleucine, leucine, phenylalanine, and tyrosine—were analyzed using isotope-dilution liquid chromatography–mass spectrometry (ID-MS). For minimizing measurement bias and uncertainty overestimation, whole spots with 50 μL of whole blood were adopted in the certification. The between-spot homogeneities by whole spot sampling were lower than 2.1%. The relative expanded uncertainties of the six amino acids in the developed DBS CRMs were lower than 5.7% at 95% confidence. The certified values are traceable to SI through both gravimetric preparation and the primary method in certification, ID-MS. Comparison among DBS testing laboratories revealed discrepancies between the whole spot and disc sampling methods. The actual sampling volume was accurately estimated by weighing, which revealed the possibility of underestimation in routine DBS testing. The candidate CRMs can support the standardization of DBS testing for amino acids through the qualification and validation of many kinds of measurement procedures to compensate the measurement bias caused by matrix-specific sampling error.

Certification of Ca, Mg, and K in human plasma reference material using isotope dilution inductively coupled plasma mass spectrometry

AbstractHuman plasma is an extracellular fluid containing elements that play an essential role in maintaining physiological balance and are therefore indispensable for human survival. A human plasma certified reference material (CRM) was developed for the accurate analysis of elements in clinical samples. Mass fractions of Ca, Mg, and K in the human plasma CRM were determined using isotope dilution inductively coupled plasma mass spectrometry. The certified values and associated expanded uncertainties obtained for Ca, Mg, and K were (74.77 ± 0.90), (15.95 ± 0.14), and (125.1 ± 5.4) mg/kg, respectively. The unit‐to‐unit variations in the elemental mass fractions evaluated from 12 units showed relative standard deviations of <1.7%, demonstrating sufficient homogeneity as the candidate CRM. A stability assessment was conducted and no noticeable changes were observed after long‐term storage for 21 months at −70 °C as well as short‐term transportation for 7 days at room temperature.

Simplified process of candidate certified reference material development for the analysis of Andrographis paniculata derived therapeutics

Rapid procedure for the separation of andrographolide from Andrographis paniculata leaves was developed to furnish a certified reference material (CRM). Andrographolide is the principle compounds, which attributed to define the quality and efficacy of A. paniculata derived medicinal products. First time, authentication, sample preparation, homogeneity, storage stability, trueness and reliability assignment by uncertainty measurement of andrographolide by high performance liquid chromatography (HPLC) were illustrated in the present paper. The impurity in the samples due to residual moisture, inorganic and organic was determined by Karl-Fisher titration, thermogravimetric analysis, and differential scanning calorimetry methods, respectively. Following the EURACHEM/CITAC guide of CG-4 statistical procedure, the certified value and corresponding expanded uncertainty of andrographolide CRM was assigned to be 99.33% ± 0.02% (k = 2) with one year stability (p < 0.05). This andrographolide CRM can be used for accurate measurement; quality controls assays and establish meteorological traceability to meet the technical requirement ISO-17025:2017.

Certification of Amyloid-Beta (Aβ) Certified Reference Materials by Amino Acid-Based Isotope Dilution High-Performance Liquid Chromatography Mass Spectrometry and Sulfur-Based High-Performance Liquid Chromatography Isotope Dilution Inductively Coupled Plasma Mass Spectrometry.

The use of amyloid-beta (Aβ) biomarkers could contribute to an early diagnosis of Alzheimer's disease (AD); however, there are still large variations among results from different assays. This variability can be overcome by standardization of those assays through the use of certified reference materials (CRMs) and the establishment of a traceability chain. In this study, Aβ40 (GBW09874) and Aβ42 (GBW09875) solution CRMs with the certified values and uncertainties of 7.58 ± 0.30 and 7.62 ± 0.30 μg g-1 were developed with high-purity Aβ as raw materials. For the first time, isotope dilution high-performance liquid chromatography mass spectrometry (ID-LC-MS) and high-performance liquid chromatography isotope dilution inductively coupled plasma mass spectrometry (HPLC-ID-ICP-MS) strategies were employed to certify the candidate Aβ solution CRMs. The two candidate CRMs showed good homogeneity, and good stability was also demonstrated for at least 5 days at -20 °C and 14 months at -70 °C. These CRMs are primarily intended to be used for value assignment to secondary calibrators or CRMs with a clinical matrix, which will help in early diagnosis of AD.

Environmental monitoring of total mercury content in different marine compartments after cold vapor generation and inductively coupled plasma mass spectrometry determination

Analytical procedure for environmental and seafood safety monitoring of total mercury content, based on the external calibration methodology and cold vapor generation inductively coupled plasma mass spectrometry determination, was validated according to the requirements of international guidelines and standards. Marine samples with varying levels of mercury: marine biota, marine sediment and costal sea waters were used in this study. A full validation approach in line with the ISO/IEC 17025 and Eurachem guidelines was followed. With this in mind, blanks, selectivity, working range (1.2 ng kg−1 to 240 ng kg−1), linearity (0.9991), recovery (97 % to 103 %), repeatability (< 2.5 %), intermediate precision (< 3.5 %), limit of detection (0.72 ng kg−1) and limit of quantification (1.10 ng kg−1) were systematically assessed. The uncertainty of measurement results was estimated according to the recommendations of the Guide to the expression of Uncertainty in Measurement. The relative expanded uncertainty was in the range between 14.2 % and 15.8 % (k = 2) for marine biota and sediment samples, respectively, and rise up to 32.8 % (k = 2) on the total mercury mass fraction in costal seawater at the level of 5 ng kg−1. The major contribution to the combined uncertainty was found to come from the uncertainty associated with the correction for recovery and procedural blanks. The performance of validated procedure was demonstrated by its application to the low-level Hg content seawater samples, received in the frame of Proficiency Testing Scheme, conducted by the Quality Assurance of Information in Marine Environment Monitoring in Europe. The result for total mercury content in marine sediment IAEA-475 was used as the contribution of the IAEA in the characterization campaign of the IAEA-475 candidate certified reference material. The demonstrated SI traceability of obtained results is a prerequisite for their further comparisons with monitoring data obtained in different place and time.

Simultaneous speciation analysis of mercury in marine origin samples by high performance liquid chromatography and species - specific isotope dilution inductively coupled plasma mass spectrometry

Species-specific isotope dilution inductively coupled plasma quadrupole mass spectrometry reference method and high-performance liquid chromatography were applied for simultaneous determination of mercury species in marine samples. Different extraction protocols for mercury species were tested and evaluated. It was found that after exposure to microwave energy the inorganic mercury (iHg) and methyl mercury (MeHg) can be completely extracted from marine biota sample by 4.5 mol L−1 HCl as well as by 3.0 mol L−1 HNO3 and further separated with HPLC. The obtained results for iHg and MeHg in the IAEA-461 certified reference material (CRM) clam Gafrarium tumidum biota sample were as follow: (341 ± 21) μg kg−1 or (338 ± 15) μg kg−1 and (61.3 ± 2.2) μg kg−1 (recovery of (98.4 ± 3.6) %) or (61.1 ± 2.8) μg kg−1 (recovery of (98.1 ± 4.6) %), respectively when diluted HCl or HNO3 were applied in the extraction step. However, these protocols are not applicable to marine sediment samples, where the iHg is the predominant form. Therefore, one selective extraction procedure for MeHg from sediment was applied to make possible its separation and quantification. This procedure consisted of series of subsequent extraction steps at room temperature: first with a mixture of H2SO4/KBr/CuSO4, next with organic solvent dichloromethane and as a final step one back-extraction with Na2S2O3 solution. The combination of the selective extraction with the ID calibration approach was found to be an efficient way for the simultaneous determination and separation of both MeHg and iHg species in one chromatographic run. The proposed procedure was successfully used for the determination of mercury species in the IAEA-405 sediment CRM and further applied for the simultaneous measurements of iHg and MeHg in a candidate CRM – the IAEA-475 sediment sample. The determined mass fractions were (30.1 ± 1.6) μg kg−1 and (196 ± 8) ng kg−1 for iHg and MeHg, respectively. The difference between obtained in this study results and those from the reference IAEA-475 values was 1.5% and further validated the developed in this study analytical methodology. To the best of our knowledge, this selective procedure for MeHg extraction was never used before for the simultaneous quantification of mercury species in marine sediment.

Traceable and accurate quantification of iron in seawater using isotope dilution calibration strategies by triple quadrupole ICP-MS/MS: Characterization measurements of iron in a candidate seawater CRM

Accurate quantification of iron is crucial for the characterization of a candidate seawater certified reference material. Isotope dilution mass spectrometry as a primary method is one of the option for this purpose. In this study, triethylamine (TEA) assisted Mg(OH)2 co-precipitation is developed and combined with isotope dilution mass spectrometry for the characterization of candidate certified reference material, UME CRM 1206. The analytical protocol of co-precipitation was optimized to achieve reproducible yields of ~100% Fe with high salt rejection (residual salinity ~0.003%). Three calibration strategies, single, double and triple isotope dilution mass spectrometry (IDMS) coupled with co-precipitation were investigated in terms of metrological aspects. The determination of Fe was carried out by ICP-MS with tandem configuration (ICP-MS/MS) using NH3/He cell gas which was optimized for the minimum background equivalence concentration to be able to measure highly accurate 56Fe/57Fe ratio with the highest possible sensitivity. The background corrected typical procedural blank levels and limit of detection calculated based on three times the standard deviation of blanks were found as 9 ng/kg (0.16 nM) and 11 ng/kg (0.20 nM) for single IDMS, respectively. Trueness of the developed analytical methods has been demonstrated using CASS-6 and NMIA MX014 with complete and very precise recoveries. SI-traceable values with combined uncertainty (k = 2) were obtained for single, double and triple IDMS as (12.70 ± 0.16) ng/g, (12.728 ± 0.084) ng/g and (12.732 ± 0.062) ng/g, respectively. The relative combined uncertainty for each approach was calculated as 0.64%, 0.33% and 0.24%, respectively.

Challenges on Production of a Certified Reference Material of Ochratoxin A in Roasted Coffee: A Brazilian Experience

Abstract Background: Coffee is an important agricultural commodity with technical barriers for exportation because of possible contamination with ochratoxin A (OTA), a mycotoxin nephrotoxic and carcinogenic. The maximum limit for OTA in roasted coffee is 5.0 μg/kg in the European Union and 10 μg/kg in Brazil, and the use of certified reference materials (CRM) is required for reliable measurements. Objective: This paper describes the development of a candidate CRM of OTA in roasted coffee following the requirements of ISO 17034 and ISO Guide 35. Methods: A primary method of isotope dilution MS was developed and validated using (13C20)-OTA as internal standard. The sample preparation was based on AOAC Official Methods of AnalysisSM using immunoaffinity column. Results: The linear working range is 2.0–15.0 μg/kg, with recoveries of 92.2–110.8% and relative SDs lower than 12.4%. The method was successfully applied to the feasibility study, which defined the procedure for preparation of a large batch around 5 μg/kg. It was produced by spiking blank roasted coffee with OTA standard, mixing and filling in amber flasks with 50 g of coffee, and storing at −80°C. The homogeneity study showed an acceptable degree of heterogeneity of 1.44%, and the short-term-stability study defined the conditions for transportation as maximum temperature of 50°C up to 28 days. Conclusions: These results show that certification is possible. Highlights: The long-term stability study at −20°C is in progress, and the characterization will be conduzed by a interlaboratory comparison. This material will be an important tool for QC in laboratories.

First Indonesian proficiency testing using reference value from isotope dilution mass spectrometry method for benzoic acid, methyl paraben, and n-butyl paraben in sweet soy sauce

Research Center for Chemistry-Indonesian Institute of Sciences (RCChem-LIPI) has successfully organized proficiency testing (PT) for the determination of benzoic acid, methyl paraben, and n-butyl paraben in sweet soy sauce. This is the first PT in Indonesia that uses reference values derived from a primary method gas chromatography–isotope dilution mass spectrometry method. Twenty participants from four economies registered in this PT. This paper presents the procedures in the preparation of the PT samples, the homogeneity and stability checks, as well as in the determination of the assigned values. The test samples were a candidate certified reference material prepared in accordance with ISO 17034 and ISO Guide 35. The performance evaluation of participants’ results was conducted in accordance with ISO 13528 by using the PROLab™ software. This paper also discusses the approaches taken in the calculation of the performance’s scores which were set according to the criterion required in homogeneity check, stability check, and limit of uncertainty.

Certification and stability assessment of recombinant human growth hormone as a certified reference material for protein quantification

A certified reference material (CRM) for the quantification of protein, essential to manage quality control and quality assurance in protein-related works, has been developed. Amino acid analysis with conventional acid hydrolysis and isotope dilution HPLC-MS was used to establish an SI-traceable absolute protein quantification method using recombinant human growth hormone (hGH) as a model protein. The certification method was verified by comparative studies between 1) different methods of protein quantification based on microwave-assisted hydrolysis, and 2) different labs as part of the Asian Collaboration on Reference Material project with Japan, China, and Korea. Certification, evaluation of measurement uncertainty, homogeneity testing, and stability testing were carried out, after which the candidate CRM for hGH quantification was successfully certified with excellent agreement within the certified value in the two comparative studies. Although the quantification value of hGH by amino acid analysis showed good robustness in various conditions, results of intact protein analysis showed degradation profiles in temperatures higher than 4 °C. Consequently, storage and dissemination conditions should be set in accordance with stability tests. Based on the results, this method is believed to be suitable for accurate quantification of hGH. Additionally, it can also be used as a guide to preparation of CRM, and instructions for quality management of protein work for other similar proteins.

Challenges on Production of a Certified Reference Material of Ochratoxin A in Roasted Coffee: A Brazilian Experience.

Background: Coffee is an important agricultural commodity with technical barriers for exportation because of possible contamination with ochratoxin A (OTA), a mycotoxin nephrotoxic and carcinogenic. The maximum limit for OTA in roasted coffee is 5.0 μg/kg in the European Union and 10 μg/kg in Brazil, and the use of certified reference materials (CRM) is required for reliable measurements. Objective: This paper describes the development of a candidate CRM of OTA in roasted coffee following the requirements of ISO 17034 and ISO Guide 35. Methods: A primary method of isotope dilution MS was developed and validated using (13C20)-OTA as internal standard. The sample preparation was based on AOAC Official Methods of AnalysisSM using immunoaffinity column. Results: The linear working range is 2.0-15.0 μg/kg, with recoveries of 92.2-110.8% and relative SDs lower than 12.4%. The method was successfully applied to the feasibility study, which defined the procedure for preparation of a large batch around 5 μg/kg. It was produced by spiking blank roasted coffee with OTA standard, mixing and filling in amber flasks with 50 g of coffee, and storing at -80°C. The homogeneity study showed an acceptable degree of heterogeneity of 1.44%, and the short-term-stability study defined the conditions for transportation as maximum temperature of 50°C up to 28 days. Conclusions: These results show that certification is possible. Highlights: The long-term stability study at -20°C is in progress, and the characterization will be conduzed by a interlaboratory comparison. This material will be an important tool for QC in laboratories.