Ochratoxin A case study: Establish metrological traceability of mycotoxin measurements using certified reference materials

For the US Food and Drug Administration's (FDA) mycotoxin program, the desired application of certified reference materials (CRMs) is primarily for validating methods for the determination of mycotoxins in foods and establishing metrological traceability of measurement results generated by such validated methods. The latter has been an important but insufficiently addressed challenge due to the lack of appropriate protocols and CRMs. Taking advantage of two recently available mycotoxin CRMs, OTAN-1 and Standard Reference Material (SRM) 1565, a protocol was developed for systematically examining uncertainty and establishing metrological traceability of measurement results of ochratoxin A (OTA) in corn through a series of calibration operations using the two CRMs. Instrument and method calibrations were performed using OTAN-1 and SRM 1565. The OTA value and its standard and expanded (k = 2, approximately 95% confidence) uncertainties were estimated from the calibration data and documented. These results demonstrate that the major contributing source of uncertainty is the sample matrix, highlighting the important role of the certified matrix reference material in method calibration. The value of OTA (38.5 ± 7.2 µg/g; 95% confidence interval) in the incurred sample was metrologically traceable to the International System of Units through two CRMs using the multi-laboratory validated liquid chromatography–mass spectrometry FDA compendial method. In addition, an alternative estimation of uncertainty was conducted using a one-point calibration, resulting in comparable uncertainty.     

Optimization of a solid phase extraction and hydrophilic interaction liquid chromatography–tandem mass spectrometry method for the determination of metformin in dietary supplements and herbal medicines

Metformin is one of the most common adulterants found in anti-diabetic dietary supplements and herbal medicines. An analytical method based on hydrophilic interaction chromatography (HILIC)/tandem mass spectrometry was developed and validated for the determination of metformin in dietary supplements and herbal medicines. Sample preparation involved solid phase extraction of the analyte using Plexa PCX cartridges, and cleanup by a primary–secondary amine (PSA) adsorbent for minimization of the matrix effect. Chromatographic separation was performed on a HILIC column using a mixture of 0.025 mol/L ammonium formate (pH 3) and acetonitrile (18:82, v/v) as the mobile phase at flow rate of 0.25 mL/min. The method was validated in four matrices (capsules, honeyed pills, tablets and oral solutions). The method had a good linear range (5–500 ng/mL), and the overall precision and accuracy ranged from 2.2% to 9.9% and 4.6% to 11.3%, respectively.     

基于蒙特卡洛法评定花生油脂肪酸组成的不确定度

采用蒙特卡洛法(monte carlo method,简称MCM)评定气相色谱法测定花生油脂肪酸组成的测量不确定度。结果表明:花生油主要脂肪酸组分C16:0的不确定度为10.78%±0.23%,C18:0的不确定度为3.52%±0.08%,C18:1n9c的不确定度为45.75%±0.58%,C18:2n6c的不确定度为32.10%±0.52%,C22:0的不确定度为2.84%±0.07%,MCM法评定测量不确定度,避免对测量模型求偏导数等复杂的测量推导及不确定度的A类评定和B类评定过程,利用MCM软件可简便快速地求出花生油二十一种脂肪酸组分测量的不确定度,该法为提高实验室不确定度评定能力提供了技术支撑。     

Analysis of non-fat soybean powder for the mass fractions of three elements: Copper and zinc by isotope dilution ICP–MS and calcium by ICP–AES

Isotope dilution inductively coupled plasma–mass spectrometry with “approximate match” approach was developed for the measurement of mass fractions of Cu and Zn. An “approximate match” (within 10%) was made between calibration and sample blends. The method was validated against two certified reference materials, namely NIST SRM 1515 (Apple Leaves) and IAEA-359 (Cabbage). Inductively coupled plasma-atomic emission spectrometry with bracketing technique was developed for the measurement of mass fraction of Ca. NIST RM 8412 (Corn Stalk) was employed for method validation purposes. The validated methods were applied to the measurement of Cu, Zn and Ca in a sample of non-fat soybean pertaining to the international comparison CCQM-P64. The results submitted for the comparison were in excellent agreement with the mean values of all participants’ results. Relative standard uncertainties of about 4% (k = 2, which gives a level of confidence of approximately 95%) were achieved in the measurement of the three elements concerned.     

超高效液相色谱-串联质谱法测定凉茶中对乙酰氨基酚的不确定度评定

目的 采用超高效液相色谱-串联质谱法(UPLC-MS/MS)测定饮料中对乙酰氨基酚的含量,进行不确定度分析。方法 依据BJS 201713《饮料、茶叶及相关制品中对乙酰氨基酚等59种化合物的测定》,采用超高效液相色谱-串联质谱法(UPLC-MS/MS)测定对乙酰氨基酚的含量,并构建不确定度评定的模型,分析不确度分量的来源,对各分量进行不确定度评定。结果 饮料中对乙酰氨基酚含量为0.406mg/kg,其测量扩展不确定度为U=0.022mg/kg,k=2。结论 该研究可为实验室饮料中对乙酰氨基酚含量的测定结果质量提供参考,其中标准溶液配制、标准曲线拟合、重复性实验是标准不确定度的重要影响分量。     

An Applicable Strategy for Improvement Recovery in Simultaneous Analysis of 20 Pesticides Residue in Tea

It is important to have a reliable method to analyze pesticides in tea, a beverage commonly consumed in Iran. A validated method was developed for the determination of 20 pesticides in tea based on QuEChERS sample preparation and capillary gas chromatography‐quadrupole mass spectrometry in selective ion monitoring mode (GC‐MS/SIM) using triphenyl methane (TPM) solution as an internal standard. We used fortified, extracted, and cleaned‐up tea samples instead of calibration standards for quantitation, which substantially reduced adverse matrix‐related effects and negative recovery affected by graphite carbon black (GCB) on pesticide analysis. The recovery of pesticides at 3 concentration (40, 60, and 240 ng/g) ranged from 79.5% to 111.4% (n = 3). The method had acceptable repeatability with RSDr < 20%. The limits of quantification (LOQ) for all pesticides were ≤20 ng/g. The analytical results of the proposed method were in good agreement with proficiency test results (FAPAS, 19116). The recoveries and repeatabilities were in accordance with the criteria set by SANCO Guideline. The validated method was suitable for the analysis of pesticides in tea.     

电感耦合等离子体质谱法测定蒙脱石中铅和砷含量的不确定度评定

目的：对电感耦合等离子体质谱法（ICP-MS法）测定蒙脱石中有害元素铅（Pb）和砷（As）含量的不确定度进行评定。方法：采用硝酸回流提取—电感耦合等离子体质谱法测定蒙脱石中Pb和As的含量。通过分析测定流程,找出不确定度的来源,并计算各不确定度分量和合成不确定度、扩展不确定度。结果：ICP-MS法测定蒙脱石中铅和砷含量的扩展不确定度为Pb 0.109mg?Kg-1,As 0.020mg?Kg-1。结论：建立的方法科学合理,为正确评定ICP-MS法测定蒙脱石中铅和砷含量的不确定度、合理使用测定数据提供了重要参考。     

Toxic metal content in 52 frequently prescribed herbal medicines on the Korean market

This study was conducted to investigate the toxic metal content (Pb, As, Cd and Hg) of 52 frequently prescribed herbal medicines and to identify herbal medicines that exceed the Korea Food and Drug Administration (KFDA) maximum limits. A total of 3534 samples, including 1966 domestic samples and 1568 imported samples, were analysed using an Inductively Coupled Plasma-Mass Spectrometer (ICP-MS). Total amounts of Pb, As, Cd and Hg were significantly different between domestic (0.63 mg kg^?1) and imported (0.81 mg kg^?1) medicines (p < 0.05). Among the 52 kinds of samples, 4 kinds of herbs required quality control for Pb and 12 kinds of herbs required quality control for Cd. No sample contained As and Hg above the limits.     

Quantification of cadmium and lead in offal by SF-ICP-MS: Method development and uncertainty estimate

Offal of bovine, ovine and porcine are able to accumulate potentially toxic heavy metals, such as Cd and Pb, posing a risk for human health. For this reason, the Commission Regulation no. 466/2001 provided the maximum admitted levels for these metals in this kind of matrix (Cd, 500 ng g⁻¹ in all kinds of offal; Pb, 500 ng g⁻¹ in liver and 1000 ng g⁻¹ in kidney). A method based on sector field inductively coupled plasma mass spectrometry for Cd and Pb quantification in calf liver was developed and further applied to offal of different animals. The uncertainty of measurements was calculated according to the Eurachem/Citac Guide. The method LoDs and LoQs were 3.5 and 11 ng g⁻¹ for Cd, and 2.0 and 6.0 ng g⁻¹ for Pb. The repeatability and the intra-laboratory reproducibility showed relative standard deviations equal to 2.25% and 1.99% for Cd and 1.49% and 6.55% for Pb. Relative expanded uncertainties at the mean value in calf liver were 4.74% for Cd and 13.8% for Pb. In Italian offal the following concentration intervals were found (in ng g⁻¹): (i) Cd: calf, from <3.5 in spleen and lung to 96.4 in kidney; lamb, <3.5 in all offal; pig, 114 in liver and (ii) Pb: calf, from 4.03 in lung to 31.8 in liver; lamb, from 4.71 in heart to 279 in liver; pig, 9.19 in liver.     

Headspace gas chromatography–mass spectrometry determination of alkylpyrazines in cocoa liquor samples

This paper proposes a fast, accurate, and quantitative method for alkylpyrazines determination in cocoa liquors samples using gas chromatography with ion trap mass spectrometry detection (GC/IT-MS) and on-line headspace (HS) sample introduction. The optimization of the experimental conditions of the headspace gas chromatography–mass spectrometry (HS–GC–MS) system was carried out using the univariate method, aiming to find a compromise between time of the analysis, sensitivity and pyrazine resolution in a Carbowax (AV-WAX) fused silica capillary column. The procedure was validated through accuracy and precision studies. The results showed a highly satisfactory accuracy rate for the method with a relative standard deviation (RSD) during 1 day and between days of <5.0% (n = 5). On the other hand, the recovery percentages (94–99%) were quantitative in all cases, with a RSD of <4.0% (n = 5). Finally, the use the on-line headspace extraction step simultaneously with the analytes separation and detection of the previous sample injection increased the analysis frequency to 3 samples/h. A total of 120 cocoa liquor samples were analyzed.     

电感耦合等离子体-质谱法直接测定果汁中砷、铅

采用微量进样、在线添加内标115In、使用动态反应池的方法,建立电感耦合等离子体质谱(inductively coupled plasma-mass spectrometry,ICP-MS)法直接测定果汁中砷、铅的方法。结果表明：方法检出限砷和铅均小于2.000μg/L,不同加标量下的As和Pb回收率分别在95.69%～105.45%和100.98%～105.14%之间,结果分析表明,测定值(0.0875μg/g)与参考值(0.085μg/g)吻合,As、Pb相对标准偏差分别为2.50%、2.94%。将比对样品经微波消解后测定结果与该直接测定方法测定结果比较无显著差异,表明该方法简便、快速、准确,完全能够满足果汁中砷、铅的检测要求。     

Method validation for the determination of total mercury in fish muscle by cold vapour atomic absorption spectrometry

A method was validated for the determination of total Hg in fish muscle using continuous flow cold vapour atomic absorption (CVAAS) after microwave digestion in closed vessels. The method was validated according to European Union Regulations 333/2007 and 657/2002, considering the maximum level for the metal in fish, established by European Union regulation 1881/2006. The procedure for determining linear range, selectivity, recovery, precision, trueness, decision limit (CCα), detection capability (CCβ), measurement uncertainty and robustness of the method is reported. The results of the validation process demonstrate the method fulfils the provisions of the Commission Regulation. The selectivity study indicated that there was no matrix effect on the calibration curve between the concentration range of 1.0 and 30.0 μg Hg l(-1). The mean recovery calculated at six levels of fortification was in the range of 94-104%. The limit of detection (LOD) and limit of quantification (LOQ) values were 4.90 and 15.7 μg kg(-1), while the CCα and CCβ values were 0.517 and 0.533 mg kg(-1), respectively, for the maximum contaminant level of 0.500 mg kg(-1). The relative expanded measurement uncertainty of the method was 0.055 mg kg(-1). The method was not affected by slight variations of some critical factors (ruggedness minor changes) as sample mass and volume of the HNO(3) and H(2)O(2) used in the digestion step. The method allowed accurate confirmation analyses of the CRM DORM 3. In fact, the Z-scores attained in a proficiency test round were well below the reference value of 2.0, proving the excellent performance of the laboratory.     

Development and validation of a front-face fluorescence spectroscopy-based method to determine casein in raw milk

A front-face fluorescence spectroscopy (FFFS)-based method for measuring casein in raw milk was developed and validated. Calibration samples (n_cal = 20) with different casein contents (0.36–3.7%) were prepared by mixing ultrafiltered retentate (2 × concentrate) and permeate at different ratios. A principal component regression model was developed using the calibration fluorescence spectra, showing a useful prediction power based on a residual prediction deviation of 3.1. The calibration model was independently validated using 20 raw milk samples. The FFFS-based method showed a root mean square error of prediction of 0.15% and 6.7% relative prediction error. A larger sample size should be included in the future to further validate the method and potentially implement it for routine measurement of casein levels in raw milk.     

Investigation of targeted pyrrolizidine alkaloids in traditional Chinese medicines and selected herbal teas sourced in Ireland using LC-ESI-MS/MS

Publications linking hepatotoxicity to the use of herbal preparations are escalating. Herbal teas, traditional Chinese medicines (TCMs) and dietary supplements have been shown to contain pyrrolizidine alkaloids (PAs). Acute PA toxicosis of the liver can result in sinusoidal-obstruction syndrome, also known as veno-occlusive disease (VOD). This paper describes a sensitive and robust method for the detection of targeted PAs and their N-oxides (PANOs) in herbal products (selected herbal teas and TCMs) sourced within Ireland. The sample preparation includes a simple acidic extraction with clean-up via solid-phase extraction (SPE). Sample extracts were accurately analysed by using LC-ESI-MS/MS applying for the first time a pentafluorophenyl (PFP) core-shell column to the chromatographic separation of PAs and PANOs. The method was validated for selectivity, taking into consideration matrix effects, specificity, linearity, precision and trueness. Limits of detection (LOD) and limits of quantitation (LOQ) were quantified for all PAs and PANOs ranging from 0.4 to 1.9 µg kg^?1 and from 1.3 to 6.3 µg kg^?1, respectively. In this study 10 PAs and four PANOs were targeted because they are commercially available as reference standards. Therefore, this study can only report the levels of these PAs and PANOs analysed in the herbal teas and TCMs. The results reported represent the minimum levels of PAs and PANOs present in the samples analysed; commercially available herbal teas (n = 18) and TCMs (n = 54). A total of 50% herbal teas and 78% Chinese medicines tested positive for one or more PAs and/or PANOs included within this study, ranging from 10 to 1733 and from 13 to 3668 µg kg^?1, respectively.     

Evaluation of an analytical methodology using QuEChERS and GC-SQ/MS for the investigation of the level of pesticide residues in Brazilian melons

A multiresidue method based on the sample preparation by modified QuEChERS and detection by gas chromatography coupled to single quadruple mass spectrometers (GC-SQ/MS) was used for the analysis of 35 multiclass pesticides in melons (Cucumis melo inodorus) produced in Ceara-Brazil. The rates of recovery for pesticides studied were satisfactory (except for the etridiazole), ranging from 85% to 117% with a relative standard deviation (RSD) of less than 15%, at concentrations between 0.05 and 0.20 mg kg⁻¹. The limit of quantification (LOQ) for most compounds was below the MRLs established in Brazil. The combined relative uncertainty (Uc) and expanded uncertainty (Ue) was determined using repeatability, recovery and calibration curves data for each pesticide. Analysis of commercial melons samples revealed the presence of pesticides bifenthrin and imazalil at levels below the MRLs established by ANVISA, EU and USEPA.     

Validation of a gas chromatography-electron capture detection of T-2 and HT-2 toxins in Chinese herbal medicines and related products after immunoaffinity column clean-up and pre-column derivatization

A sensitive, reproducible and accurate gas chromatography-electron capture detection (GC-ECD) method was developed for simultaneous determination of T-2 and HT-2 toxins in Chinese herbal medicines (CHMs) and related products after immunoaffinity column (IAC) clean-up and pre-column derivatization with N-heptafluoro-butyryl imidazole (HFBI). Then, gas chromatography-spectrometry spectrometer (GC-MS) was applied to confirm the positive results and interfering peaks. The limits of detection (LODs) for T-2 and HT-2 toxins were 1.88 and 0.47 ng/g, and the recoveries for different CHMs ranged from 89.2% to 99.1% with relative standard deviation (RSD) <6.0% for T-2 and from 85.9% to 99.0% with RSD <8.8% for HT-2 toxin, respectively. The validated method was successfully applied for the determination of T-2 and HT-2 toxins in 89 Chinese herbal medicines and 10 related products from various sources, where it was found that T-2 and HT-2 toxins were not detected in any of the tested samples. These results were reliable by confirmation using GC-MS. Some unknown peaks were interfering peaks not the target toxins.     

Optimisation and validation of a quantitative and confirmatory LC-MS method for multi-residue analyses of β-lactam and tetracycline antibiotics in bovine muscle

A multi-residue method for the determination of the β-lactam antibiotics ampicillin, cefazolin, cloxacillin, dicloxacillin, nafcillin, oxacillin, penicillin G, penicillin V and the tetracyclines chlotetracycline, tetracycline and oxytetracycline was optimised and validated in bovine muscle. The method is based on the extraction of the residues from muscle using water/acetonitrile (2/8, v/v) with subsequent use of dispersive solid-phase C18 and hexane for purification. Extracts were analysed using ultra-performance liquid chromatography (UPLC-MS/MS) coupled with the mass spectrometer in positive electrospray ionisation mode (ESI+) for all analytes. The method was validated according to the requirements of European Commission Decision 2002/657/EC. The validation results were obtained within the MRL range of 0-1.5 of the MRL, with recoveries varying from 90% to 110% and CV < 20% (n = 54), except for cloxacillin, dicloxacillin and nafcillin. However, matrix interference was observed. The decision limit (CCα) ranged from 10% to 15% of the MRL. The uncertainty measurement was estimated based on both bottom-up and top-down strategies and the uncertainty values were found to be lower than 20% of the MRL. The method has a simple extraction procedure whereby analytes are separated with reasonable resolutions in a single 11-min chromatographic run. According to the validation results, this method is suitable for monitoring β-lactams and tetracyclines according to National Program for Residue and Contaminant Control - Brazil (NPRC-Brazil) in bovine muscle.     

Solid‐phase extraction of lead and copper on a polyhydroxybutyrate‐b‐polydimethyl siloxane (PHB‐b‐PDMS) block copolymer disc and flame atomic absorption spectrometric determination of them in water and food samples

A separation and preconcentration procedure based on solid‐phase extraction of Cu(II) and Pb(II) as their Sudan III chelates on polyhydroxybutyrate‐b‐polydimethyl siloxane (PHB‐b‐PDMS) block copolymer disc has been established. Various analytical parameters including pH, amount of Sudan III, eluent type and volume, sample volume, flow rates were optimised. The effects of concomitant ions on the retentions of the analyte ions on PHB‐b‐PDMS disc were also examined. The detection limits for Cu(II) and Pb(II) were found to be 2.3 and 4.9 μgL^?1, respectively. Preconcentration factor was found to be sixty. Relative standard deviation was found to be 5%. The accuracy of the method was confirmed by analysing certified reference materials (TMDA 54.4 fortified lake water and NIST 1515 Apple leaves). Optimised method was applied to determine the analyte ions in various water and food samples from Turkey.     

Validation of a Method for Analysis of Aroma Compounds in Red Wine using Liquid–Liquid Extraction and GC–MS

An analytical method for determination of volatile composition of wines using sample preparation by liquid–liquid extraction and gas chromatography coupled to mass spectrometry for separation and detection has been developed and validated. Extraction of volatile compounds was performed in dichloromethane, and 1-octanol was added as an internal standard. Kékfrankos red wine produced in Villány wine region in Hungary was used as a model wine for testing and validation of the method. The developed method allowed satisfactory determination of 33 volatile compounds in the wines. Compounds analyzed include alcohols, esters, lactones, fatty acids, furans, and nitrogen compounds. The calibration curves of the four reference compounds used (2-phenyl ethanol, ethyl nonanoate, butyrolactone, and tyrosol) were linear in all cases with correlation coefficients (R ²) ranging from 0.9951 to 0.9992. The accuracy of the method was checked with a standard addition method (recovery 92.2–103 %), showing good repeatability and reproducibility (RSD?<?10 %).     

液相色谱-串联质谱法检测中兽药中非法添加氟喹诺酮类药物含量的不确定度评估

目的评估液相色谱-串联质谱法检测中兽药中非法添加氟喹诺酮类药物含量的不确定度。方法建立液相色谱-串联质谱法检测中兽药(肥猪散、健胃散、银翘散)中非法添加氟喹诺酮类药物的含量测定方法,根据JJF1059.1-2012《测量不确定度评定与表示》的相关规定,对该方法的不确定度进行评定。结果中兽药中氟喹诺酮类药物含量分别为C(Enro)=100.90 mg/kg、C(Cip)=100.34 mg/kg、C(Norf)=100.21 mg/kg、C(Oflo)=100.61 mg/kg时,95%置信水平下,取k=2,不确定度评估结果分别表示为X(Enro)=(100.90±6.957)mg/kg;X(Cip)=(100.34±7.067) mg/kg; X(Norf)=(100.21±6.898) mg/kg; X(Oflo)=(100.61±6.957) mg/kg。结论评定结果表明,影响检测结果的主要因素为液相色谱-串联质谱仪的灵敏度、天平的精密度、操作的规范性。