A mussel tissue certified reference material for multiple phycotoxins. Part 2: liquid chromatography-mass spectrometry, sample extraction and quantitation procedures

A freeze-dried mussel tissue certified reference material (CRM-FDMT1) containing multiple groups of shellfish toxins has been prepared. Toxin groups present in the material include okadaic acid and the dinophysistoxins, azaspiracids, yessotoxins, pectenotoxins, spirolides and domoic acid. In this work, analytical methods have been examined for the characterisation of the candidate CRM. A comprehensive extraction procedure was developed, which gave good recovery (>98%) for all lipophilic toxins studied. A fast liquid chromatography–mass spectrometry (LC-MS) method was developed that separates the major toxins according to the MS ionisation mode of optimum sensitivity. Matrix effects associated with analysis of these extracts using the developed LC-MS method were assessed. Standard addition and matrix-matched calibration procedures were evaluated to compensate for matrix effects. The methods and approaches will be used for the precise characterisation of the homogeneity and stability of the various toxins in CRM-FDMT1 and for the accurate assignment of certified values. The developed methods also have excellent potential for application in routine regulatory monitoring of shellfish toxins.     

Development of bovine serum albumin certified reference material

We present the development process for National Institute of Metrology (NIM) bovine serum albumin (BSA) certified reference material (CRM). Each CRM unit contains about 200 mg of purified BSA. The moisture, ignition residue, molecular weight, and high-performance liquid chromatography (HPLC) purity were analyzed and mass spectrometry based protein identification was carried out to ensure the material was BSA. Both amino acid based isotope dilution mass spectrometry (IDMS) and a purity deduction method were selected for value assignment. The certified value was the average of the IDMS and the purity deduction result. HPLC purity analysis was used to examine the homogeneity and stability of solid BSA CRM. Fifteen units were selected for between-bottle homogeneity examination and seven subsamples from the same bottle were selected for within-bottle homogeneity examination. Statistics showed the CRM passed both the between-bottle and the within-bottle homogeneity examination. The CRM stability under storage conditions (-20 °C) was tested for 18 months and no trend was observed. Uncertainties from the balance, amino acid purity, hydrolysis, method reproducibility, homogeneity, and stability were taken into account in uncertainty evaluation. The final certified value of NIM BSA CRM is (0.963±0.038) g/g.     

Freeze-drying for the stabilisation of shellfish toxins in mussel tissue (<Emphasis Type="Italic">Mytilus edulis</Emphasis>) reference materials

Two samples of mussels (Mytilus edulis) were collected from the southwest of Ireland. One sample contained domoic acid, the other sample contained okadaic acid, dinophysistoxin-2 and azaspiracid-1, -2 and -3. Wet and freeze-dried reference materials were prepared from each of the two samples to test for differences in homogeneity, stability and extractability of the analytes in either condition. Wet materials were homogenised, aliquoted and hermetically sealed under argon and subsequently frozen at −80 °C. Dry materials were similarly homogenised but frozen in flat cakes prior to freeze-drying. After grinding, sieving and further homogenisation, the resulting powder was aliquoted and hermetically sealed. Domoic acid materials were characterised using HPLC–UV, while LC–MS was used for the determination of lipophilic toxins. The extractabilities of all phycotoxins studied were comparable for wet and freeze-dried materials once a sonication step had been carried out for reconstitution of the freeze-dried materials prior to extraction. Homogeneity was assessed through replicate analysis of the phycotoxins (n = 10), and was found to be similar for wet and freeze-dried materials, for both hydrophilic and lipophilic toxins. Water contents were determined for both wet and freeze-dried materials, and particle size was determined for the freeze-dried materials. Stability was evaluated isochronously over eight months at four temperatures (−20, +4, +20 and +40 °C). The freeze-dried material containing domoic acid was stable over the whole duration at all temperatures, while in the wet material domoic acid degraded to some extent at all temperatures except −20 °C. In freeze-dried and wet materials containing lipophilic toxins, okadaic acid, dinophysistoxin-2, azaspiracid-1 and azaspiracid-2 were stable over the whole duration at all conditions, while concentrations of azaspiracid-3 changed significantly in both materials at some storage temperatures. Figure Aliquots of freeze-dried and wet mussel tissue reference materials containing the various shellfish toxins examined in the study     

A mussel tissue certified reference material for multiple phycotoxins. Part 1: design and preparation

The development of multi-analyte methods for lipophilic shellfish toxins based on liquid chromatography–mass spectrometry permits rapid screening and analysis of samples for a wide variety of toxins in a single run. Validated methods and appropriate certified reference materials (CRMs) are required to ensure accuracy of results. CRMs are essential for accurate instrument calibration, for assessing the complete analytical method from sample extraction to data analysis and for verifying trueness. However, CRMs have hitherto only been available for single toxin groups. Production of a CRM containing six major toxin groups was achieved through an international collaboration. Preparation of this material, CRM-FDMT1, drew on information from earlier studies as well as improved methods for isolation of toxins, handling bulk tissues and production of reference materials. Previous investigations of stabilisation techniques indicated freeze-drying to be a suitable procedure for preparation of shellfish toxin reference materials and applicable to a wide range of toxins. CRM-FDMT1 was initially prepared as a bulk wet tissue homogenate containing domoic acid, okadaic acid, dinophysistoxins, azaspiracids, pectenotoxin-2, yessotoxin and 13-desmethylspirolide C. The homogenate was then freeze-dried, milled and bottled in aliquots suitable for distribution and analysis. The moisture content and particle size distribution were measured, and determined to be appropriate. A preliminary toxin analysis of the final material showed a comprehensive toxin profile.     

Development of a certified reference material for the determination of acrylamide in potato chips

A certified reference material (CRM), KRISS CRM 108-10-003, has been developed for analysis of acrylamide in potato chips, as a representative of carbohydrate-rich food cooked in high-temperature oil. The material was prepared by grinding commercially available potato chips to a paste which was then homogenized, bottled in 15-g units, and stored at ?70 °C. Certification, homogeneity and stability testing, were carried out by liquid chromatography–isotope-dilution mass spectrometry (ID-LC–MS). A single ID-LC–MS measurement was performed for each of 10 selected units for certification and homogeneity assessment. The mean measurement result for the 10 bottles, 0.455?±?0.012 mg?kg^?1, was assigned as the certified value of the CRM. The between-bottle homogeneity was 0.8% of the certified value. The within-bottle homogeneity, tested by measuring three replicate sub-samples from each of three randomly selected bottles, was similar to the between-bottle homogeneity. The stability of the CRM under storage conditions (?70 °C) was tested for 21 months and no change in the acrylamide content was observed within the measurement uncertainty. Stability of the CRM at –20 °C (storage at user’s site) and room temperature (for regular use and transportation) was also tested. Also presented is the newly designed procedure for evaluating the uncertainty of the certified value for the characterization scheme used in this study.

Development of reference materials for paralytic shellfish poisoning toxins

A project was undertaken to develop mussel reference materials that were certified for their mass fractions of saxitoxin and decarbamoyl-saxitoxin. Fifteen laboratories from various European countries participated. Three of these had major responsibility for substantial parts of the work and overall coordination of the project. The project involved 4 main activities: (1) procurement and characterization of calibrants; (2) improvement of analytical methodology; (3) preparation of reference materials, including homogeneity and stability studies; (4) 2 interlaboratory studies and a certification exercise. The joint activities resulted in 3 homogeneous and stable reference materials: 2 lyophilized mussel materials with and without naturally incurred paralytic shellfish poisoning (PSP) toxins, and a saxitoxin enrichment solution. The reference materials were certified with respect to their saxitoxin and decarbamoyl-saxitoxin content. The lyophilized mussel material with PSP toxins (CRM 542) contained <0.07 mg saxitoxin x 2HCl/kg and 1.59 +/- 0.20 mg decarbamoyl-saxitoxin x 2HCl/kg. The lyophilized mussel material without PSP toxins (CRM 543) contained <0.07 mg saxitoxin x 2HCl/kg and <0.04 mg decarbamoyl-saxitoxin x 2HCl/kg. The certified value of the saxitoxin mass fraction in the saxitoxin enrichment solution (CRM 663) was 9.8 +/- 1.2 microg/g.     

Development of hemoglobin A1c certified reference material by liquid chromatography isotope dilution mass spectrometry

We report the development of a National Institute of Metrology (NIM) hemoglobin A_1c (HbA_1c) certified reference material (CRM). Each CRM unit contains about 10 μL of hemoglobin. Both hemoglobin and glycated hemoglobin were quantitatively determined by high-performance liquid chromatography (HPLC)–isotope dilution mass spectrometry (IDMS) with synthesized VHLTPE and glycated VHLTPE as standards. The mass fraction of synthesized VHLTPE or glycated VHLTPE was also quantitatively determined by HPLC-IDMS with NIM amino acid CRMs as standards. The homogeneity and stability of the CRMs were examined with a commercial HbA_1c analyzer based on the HPLC principle. Fifteen units were randomly selected for homogeneity examination, and statistical analysis showed there was no inhomogeneity. Examination of the stability showed that the CRM was stable for at least 6 months at -80 °C. Uncertainty components of the balance, amino acid purity, hydrolysis and proteolysis efficiency, method reproducibility, homogeneity, and stability were taken into consideration for uncertainty evaluation. The certified value of NIM HbA_1c CRM was expressed as the ratio of HbA_1c to total hemoglobin in moles, and was (9.6 ± 1.9)% . The CRM can be used as a calibration or validation standard for clinical diagnostics. It is expected to improve the comparability for HbA_1c measurement in China.     

Evaluation of the stability of ethanol in water certified reference material: measurement uncertainty under transport and storage conditions

This study simulated the transport and storage conditions of ethanol in water certified reference material (CRM) produced by the Chemical Metrology Division of Inmetro—DQUIM with the purpose of estimating the measurement uncertainty related to stability. The short-term stability study was performed on five different mass fractions (w) in terms of mg ethanol/g solution of the ethanol in water CRM. The nominal values are w = 0.5, 0.9, 1.1, 3.8 and 4.6 mg/g, at temperatures of 4 and 60 °C. On the other hand, the long-term stability study was developed on four different mass fractions (nominal values): w = 0.5, 0.9, 1.1 and 4.6 mg/g, at a temperature of 20 °C. This paper will show the data from the long-term stability study that took place over 52 weeks. The method used complies with ISO Guide 35, the BCR Guideline for Feasibility Studies and ISO Guide 34. According to the statistical parameters used in both studies, the stability of ethanol in water CRM was confirmed for all of the mass fractions studied.     

A feasibility study into the production of a freeze-dried oyster reference material for paralytic shellfish poisoning toxins

Matrix reference materials are an essential component for the validation and quality control of analytical methodologies for the quantitation of marine biotoxins in shellfish. Given the potential advantages of reference materials in powder form, a study was conducted to assess the feasibility for the production of a freeze-dried oyster tissue reference material containing a range of important paralytic shellfish poisoning toxins. One bulk sample of a wet oyster tissue homogenate was generated following mass culturing of toxic Alexandrium and oyster feeding experiments. The bulk tissue was used to prepare untreated wet frozen aliquots with the remainder being freeze-dried and processed into appropriately-sized powder samples. A pre-column oxidation LC-FLD analysis was used to confirm the absence of any chromatographic artefacts resulting from the processing and to confirm acceptable homogeneity of the tissues. Excellent stability over both the short-term (1 month) and long-term (1 year) of the freeze-dried material was demonstrated as compared with the stability of the untreated wet tissue. A post-column oxidation LC-FLD method was used to confirm the absence of toxin epimerisation in freeze-dried tissues which were observed in the wet tissues. Overall the work showed the feasibility of an approach to produce a homogenous freeze-dried oyster matrix material with enhanced stability in comparison to the untreated wet tissue. The potential for use of the process for preparation of large scale production batches of a freeze-dried CRM for paralytic shellfish poisoning toxins has therefore been demonstrated.     

Reference materials for domoic acid, a marine neurotoxin

Summary Domoic acid was recognized recently [1, 2] to be a marine neurotoxin associated with shellfish harvested or cultured for use as human food. Evidence about the occurrence of domoic acid and its importance to shellfish industries is reviewed. The preparation and certification of two reference materials for the determination of domoic acid, an instrument calibration solution (DACS-1, released by the Marine Analytical Chemistry Standards Program [MACSP] in May, 1989), and a tissue reference material (MUS-1, homogenized soft tissues of Mytilus edulis, released by the MACSP in August, 1989) are described. We believe these are the first certified standards or reference materials to be available for the determination of shellfish toxins, a problem of increasing importance to aquaculturists and the seafood industry, as well as to agencies concerned with the safety of food. The tissue homogenate preparation techniques we have developed and used may be of general interest for the preparation of other tissue reference materials for the determination of other organic compounds, since the sealed, fluid homogenate samples seem acceptably stable without being continuously frozen or refrigerated.NRCC No. 31925     

Screening for multiple classes of marine biotoxins by liquid chromatography-high-resolution mass spectrometry

Marine biotoxins pose a significant food safety risk when bioaccumulated in shellfish, and adequate testing for biotoxins in shellfish is required to ensure public safety and long-term viability of commercial shellfish markets. This report describes the use of a benchtop Orbitrap system for liquid chromatography–mass spectrometry (LC-MS) screening of multiple classes of biotoxins commonly found in shellfish. Lipophilic toxins such as dinophysistoxins, pectenotoxins, and azaspiracids were separated by reversed phase LC in less than 7 min prior to MS data acquisition at 2 Hz with alternating positive and negative scans. This approach resulted in mass accuracy for analytes detected in positive mode (gymnodimine, 13-desmethyl spirolide C, pectenotoxin-2, and azaspiracid-1, -2, and -3) of less than 1 ppm, while those analytes detected in negative mode (yessotoxin, okadaic acid, and dinophysistoxin-1 and -2) exhibited mass errors between 2 and 4 ppm. Hydrophilic toxins such as domoic acid, saxitoxin, and gonyautoxins were separated by hydrophilic interaction LC (HILIC) in less than 4 min, and MS data was collected at 1 Hz in positive mode, yielding mass accuracy of less than 1 ppm error at a resolving power of 100,000 for the analytes studied (m/z 300–500). Data were processed by extracting 5 ppm mass windows centered around the calculated masses of the analytes. Limits of detection (LOD) for the lipophilic toxins ranged from 0.041 to 0.10 μg/L (parts per billion) for the positive ions, 1.6–5.1 μg/L for those detected in negative mode, while the domoic acid and paralytic shellfish toxins yielded LODs ranging from 3.4 to 14 μg/L. Toxins were detected in mussel tissue extracts free of interference in all cases.     

Reference materials characterized for impurities in uranium matrices: An overview and re-evaluation of the NBL CRM 124 series

The characterized concentrations of 24 impurity elements in New Brunswick Laboratory (NBL) Certified Reference Material (CRM) 124 were reevaluated. A provisional certificate of analysis was issued in September 1983 based upon the “as prepared” values (gravimetric mixing). The provisional certificate does not state uncertainties for the characterized values, or estimate the degree of homogeneity. Since release of the provisional certificate of analysis various laboratories have reported analytical results for CRM 124. Based upon the reported data a re-evaluation of the characterized values with an estimate of their uncertainties was performed in this work. An assessment of the degree of homogeneity was included. The overall difference between the re-evaluated values for the 24 impurity elements and the “as prepared” values from the provisional certificate of analysis is negligible compared to the uncertainties. Therefore, NBL will establish the “as prepared” values as the certified values and use the derived uncertainties from this work for the uncertainties of the certified values. The traceability of the “as prepared” values was established by the gravimetric mixing procedure employed during the preparation of the CRM. NBL further recommends a minimum sample size of 1 g of the CRM material to ensure homogeneity. Samples should be dried by heating up to 110 °C for one hour before use.     

Feasibility of gamma irradiation as a stabilisation technique in the preparation of tissue reference materials for a range of shellfish toxins

The effect of γ-irradiation on concentrations of hydrophilic and lipophilic phycotoxins has been investigated by use of HPLC–UV and LC–MS. Pure toxins in organic solvents and toxins in mussel (Mytilus edulis) tissues were irradiated at three different doses. In solution all toxin concentrations were reduced to some extent. Most severe decreases were observed for domoic acid and yessotoxin, for which the smallest dose of irradiation led to almost complete destruction. For pectenotoxin-2 the decrease in concentration was less severe but still continuous with increasing dose. Azaspiracid-1 and okadaic acid were the least affected in solution. In shellfish tissue the decrease in toxin concentrations was much reduced compared with the effect in solution. After irradiation at the highest dose reductions in concentrations were between ca. 5 and 20% for the lipophilic toxins and there was no statistical difference between control and irradiated samples for azaspiracids in tissue. Irradiation of shellfish tissues contaminated with domoic acid led to a more continuous decrease in the amount of the toxin with increasing dose. The effect of irradiation on the viability of microbial activity in shellfish tissues was assessed by using total viable counting techniques. Microbial activity depended on the type of shellfish and on the pretreatment of the shellfish tissues (with or without heat treatment). As far as we are aware this is the first investigation of the effectiveness of irradiation as a technique for stabilising tissue reference materials for determination of phycotoxins. Our results suggest that this technique is not effective for materials containing domoic acid. It does, however, merit further investigation as a stabilisation procedure for preparation of shellfish tissue materials for some lipophilic toxins, in particular azaspiracids. Chemical structures of the toxins investigated in the study     

The possibility of using beef tallow biodiesel as a viscosity reference material

This study analyses the possibility of using beef tallow biodiesel transesterified with ethanol, provided by Universidade Federal Fluminense, as a viscosity reference material for biodiesels. The quantity viscosity was measured with capillary viscometers, according to Brazilian standards, in a temperature range between 20 °C and 40 °C for characterisation, and at 40 °C for homogeneity, in short-term stability (90 days) and long-term stability (450 days). Thirty-nine samples were stored, 9 at 45 °C, 6 at 4 °C and 24 at 20 °C. The behaviour of viscosity is analysed considering the estimated uncertainty of measurements for characterisation, homogeneity and stability. The reason to study a transesterified biofuel with ethanol lies in the fact that it is easy to produce this fluid from sugarcane in Brazil.     

The preparation of a certified reference material of polar pesticides in freeze-dried water (CRM 606)

The preparation of a certified reference material of polar pesticides in freeze-dried water is described. The pesticides selected were atrazine, simazine, carbaryl, propanil, linuron, fenamiphos and permethrin which were added to 6000 litres of tap water at 50–80 μg · L^–1 (200–320 μg · L^–1 for permethrin) level in presence of NaCl (2.5 g · L^–1) prior lyophilization. After the freeze-drying process the residue was rehomogenized, filled into amber glass bottles and stored at –20?°C, +4?°C and +20?°C. All pesticides were determined by HPLC/diode array detector, except permethrin which was determined by GC/ECD. The results obtained for atrazine, simazine, carbaryl, propanil, linuron and fenamiphos showed no within- or between-bottle inhomogeneity, however the material was non-homogeneous for permethrin and therefore this was withdrawn from further studies. With respect to the stability for over one year, all pesticides were stable at –20?°C. At +4?°C all pesticides were stable for at least 9 months and at +20?°C the stability was demonstrated only during the first month of storage. The content (mass fractions) of atrazine, simazine, carbaryl, propanil and linuron in freeze-dried water (CRM 606) was certified by an interlaboratory testing and a certification campaign.     

The preparation of a certified reference material of polar pesticides in freeze-dried water (CRM 606)

The preparation of a certified reference material of polar pesticides in freeze-dried water is described. The pesticides selected were atrazine, simazine, carbaryl, propanil, linuron, fenamiphos and permethrin which were added to 6000 litres of tap water at 50–80 μg · L^–1 (200–320 μg · L^–1 for permethrin) level in presence of NaCl (2.5 g · L^–1) prior lyophilization. After the freeze-drying process the residue was rehomogenized, filled into amber glass bottles and stored at –20 °C, +4 °C and +20 °C. All pesticides were determined by HPLC/diode array detector, except permethrin which was determined by GC/ECD. The results obtained for atrazine, simazine, carbaryl, propanil, linuron and fenamiphos showed no within- or between-bottle inhomogeneity, however the material was non-homogeneous for permethrin and therefore this was withdrawn from further studies. With respect to the stability for over one year, all pesticides were stable at –20 °C. At +4 °C all pesticides were stable for at least 9 months and at +20 °C the stability was demonstrated only during the first month of storage. The content (mass fractions) of atrazine, simazine, carbaryl, propanil and linuron in freeze-dried water (CRM 606) was certified by an interlaboratory testing and a certification campaign. Received: 7 September 1998 / Revised: 13 November 1998 / Accepted: 21 November 1998     

藏药诃子质量标志物诃子酸的分离分析及标准化研究

依据GB/T 15000—2008《标准样品工作导则》的要求,研制诃子酸国家标准样品.以诃子的干燥成熟果实为原料,采用大孔吸附树脂、制备型高效液相色谱技术对标准样品进行制备,经过纯度分析、结构鉴定、均匀性检验、稳定性检验,最后进行联合定值.基于高效液相色谱(HPLC)技术,采用不同检测波长进行纯度测试,样品纯度均大于98%.基于液相-质谱(LC-MS)进行纯度分析,未发现明显杂质峰.通过紫外光谱(UV)、红外光谱(IR)、质谱(MS)、核磁共振(NMR)等技术确定其结构为诃子酸.样品均匀性良好,4℃条件下24个月内稳定性良好.定值结果确定诃子酸纯度为98.08%,在95%置信区间范围内的相对扩展不确定度为0.50%.研制出的诃子酸(GSB 11-3725-2020)国家标准样品可用于含量测定、检测方法评定、相关产品的检测与质量控制.     

加替沙星纯度标准物质研制

研制加替沙星纯度标准物质。采用质量平衡法和氢谱定量核磁法对加替沙星纯度标准物质候选物进行纯度定值,采用卡尔费休法和热重分析法准确测量了影响主成分纯度的水分含量,并考察了水分含量的稳定性。开展了加替沙星纯度定值、均匀性检验、稳定性考察以及不确定度评估。结果表明,加替沙星纯度标准物质的纯度值为93.1%,扩展不确定度为0.6%(k=2),水分含量为6.6%,特性量值均匀性良好,6个月稳定性可靠,被认定为国家二级标准物质,编号为GBW(E) 100476。研制的加替沙星纯度标准物质填补了国内空白,同时也为吸湿性较强的药物纯度标准物质研制过程中水分测定提供了参考与借鉴。     

Development of a certified reference material for cachaça: an effective material for quality assurance

Cachaça is the typical and genuine denomination for the sugar cane beverage produced in Brazil. It has an alcoholic content between 38 and 48 % by volume, at 20 °C, and is obtained from the distillation of fermented sugar cane juice with the possible addition of up to 6 g/L of sugar, expressed by sucrose. This paper aims to outline the approach taken for the certification of the mass fraction of individual alcohols and their associated uncertainties in a cachaça certified reference material (CRM) produced by the Organic Analysis Laboratory of the Chemical Metrology Division of Inmetro. The requirements of ISO Guide 31, ISO Guide 34 and ISO Guide 35 were followed for the certification. This included the assessment of the degree of homogeneity of the material, the short-term stability study to determine the transport conditions, the long-term stability study to establish the shelf life and storage conditions of the CRM and the characterization process. The certified values were determined by two analytical techniques: gas chromatography coupled to mass spectrometry and gas chromatography with flame ionization detection. In order to support the routine testing of cachaça, in helping analytical laboratories validate and standardize their methods, the certified reference material (CRM 02.1/10.003b) was developed and certified for the mass fractions of methanol (9.18 mg/100 g ± 0.82 mg/100 g), 2-butanol (4.15 mg/100 g ± 0.40 mg/100 g), 1-butanol (1.30 mg/100 g ± 0.18 mg/100 g), isobutanol (24.0 mg/100 g ± 1.0 mg/100 g) and 1-propanol (29.4 mg/100 g ± 1.3 mg/100 g) in a spiked cachaça.     

Certification of total arsenic, dimethylarsinic acid and arsenobetaine contents in a tuna fish powder (BCR-CRM 627)

Tuna fishes were collected in the Straits of Messina (Italy), were dissected and dorsal muscles minced, freeze-dried, ground and sieved. The obtained powder was stabilised by γ-irradiation and filled into brown glass bottles. The homogeneity and stability at +20 °C and +40 °C were verified with regards to the total arsenic, dimethylarsinic acid and arsenobetaine contents. Ten laboratories participated in the certification study. All participants had demonstrated beforehand their ability to produce accurate results for the As speciation in fish tissue. The certified values are: total arsenic (4.8 ± 0.3) mg/kg, arsenobetaine (52 ± 3) μmol/kg, dimethylarsinic acid (2.0 ± 0.3) μmol/kg. The material is available from the BCR since early 1998. Received: 31 March 1998 / Revised: 20 July 1998 / Accepted: 25 July 1998