茶碱标准物质的研制与定值

建立了茶碱标准物质的制备和定值方法.将市售茶碱原料纯化、干燥、混匀后.分装成500瓶样品.随机抽取其中11瓶做均匀性检验.经F检验和T检验.结果表明在95%置信范围内样品的均匀性良好;稳定性检验按短期稳定性(1月)和长期稳定性(12月)分别进行,长期稳定性结果通过了T检验,表明在20℃条件下茶碱的稳定期不少于12个月....     

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.     

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.     

黄芩素纯度标准物质的定值及其不确定度评定

为了满足食品及医药等领域的检测需求,研制了黄芩素纯度标准物质。采用液相色谱–质谱法和红外光谱法对黄芩素纯度标准物质原料定性后,利用高效液相色谱法(HPLC)和定量核磁技术(Quantitative Nuclear Magnetic Resonance Spectroscopy,QNMR)对黄芩素的纯度进行了定值,并用HPLC法对黄芩素纯度标准物质进行了均匀性检验和稳定性考察。对定值结果的不确定度进行了评价,研制的黄芩素纯度标准物质的定值结果和扩展不确定度分别为98.8%,0.8%(k=2)。     

Accurate determination of arsenic in arsenobetaine standard solutions of BCR-626 and NMIJ CRM 7901-a by neutron activation analysis coupled with internal standard method

Neutron activation analysis (NAA) coupled with an internal standard method was applied for the determination of As in the certified reference material (CRM) of arsenobetaine (AB) standard solutions to verify their certified values. Gold was used as an internal standard to compensate for the difference of the neutron exposure in an irradiation capsule and to improve the sample-to-sample repeatability. Application of the internal standard method significantly improved linearity of the calibration curve up to 1 μg of As, too. The analytical reliability of the proposed method was evaluated by k₀-standardization NAA. The analytical results of As in AB standard solutions of BCR-626 and NMIJ CRM 7901-a were (499 ± 55) mg kg⁻¹ (k = 2) and (10.16 ± 0.15) mg kg⁻¹ (k = 2), respectively. These values were found to be 15-20% higher than the certified values. The between-bottle variation of BCR-626 was much larger than the expanded uncertainty of the certified value, although that of NMIJ CRM 7901-a was almost negligible.     

甲基苯丙胺标准物质的定值与不确定度分析

为了建立法庭科学毒品检测量值溯源体系,研制了甲基苯丙胺纯度标准物质。通过红外光谱、质谱法对甲基苯丙胺样品进行定性分析,优化并建立了高效液相色谱(HPLC)、差示扫描量热法(DSC)两种定值分析方法。采用热重分析法和电感耦合等离子体质谱法测定水分和无机离子的含量。通过均匀性与稳定性检验结果表明,甲基苯丙胺标准物质的均匀性良好,稳定性至少1年,同时对其进行了不确定度评定。研制的甲基苯丙胺标准物质的纯度为99.8%,扩展不确定度为0.2%(k=2)。     

Development of a certified reference material for the determination of perfluorooctanoic acid

A certified reference material (CRM) for the determination of perfluorooctanoic acid (PFOA) has been issued as NMIJ CRM 4056-a by the National Metrology Institute of Japan, National Institute of Advanced Industrial Science and Technology (NMIJ/AIST). Purity (kg kg^?1) based on a titration method was determined by subtracting the mass fractions of impurities measured using liquid chromatography/mass spectrometry (LC/MS) from those of acids expressed as PFOA measured by a neutralization potentiometric titration. To validate an obtained result, purity based on a mass balance method was determined by subtracting the mass fractions of impurities, measured using LC/MS, Karl-Fischer titration (KFT), and vacuum evaporation, from 1 kg kg^?1. Results from both titration and mass balance methods were in agreement within the accepted limits of uncertainty. The certified purity of NMIJ CRM 4056-a was determined to be 0.959 kg kg^?1, calculated as the mean of the results obtained with the two methods. The standard uncertainty of the certified purity was evaluated from purity evaluations as well as from sample homogeneity and stability obtained from LC/MS and KFT analyses. Consequently, the expanded uncertainty was estimated to be 0.005 kg kg^?1 with a coverage factor of k = 2.     

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.     

差示扫描量热法对芝麻酚纯度标准物质的定值

建立了采用差示扫描量热法对芝麻酚纯度标准物质的定值及不确定度评价的数学模型、有效检测技术和分析方法.采用差示扫描量热法测量芝麻酚样品纯度的实验条件为升温速率3.0 K/min,称样量3.4～4.7 mg,炉内气体为静态空气.对通过均匀性检验和长期稳定性考察的芝麻酚纯度标准物质进行定值和不确定度评价,同时采用高效液相色谱...     

诱惑红溶液标准物质的研制及不确定度评定

建立了食用合成色素诱惑红溶液标准物质的制备和定值方法,研制了100 mg/L的诱惑红溶液标准物质。采用制备液相色谱对筛选的市售原料纯化,得到纯度大于99%的诱惑红纯品;通过核磁共振(1H NMR谱)和液相色谱-质谱(HPLC-LTQ/MS)准确定性分析后,利用高效液相色谱法(HPLC)对诱惑红纯物质进行纯度定值。以0.1 mol/L乙酸铵和甲醇为流动相进行等度洗脱,采用Intersil ODS-C18(250 mm×4.6 mm,5μm)进行分离,检测波长240 nm。为保证纯度测量的准确性,采用多家联合定值对诱惑红的纯度进行定值,诱惑红纯物质的定值纯度为99.61%(λ=240 nm)。诱惑红溶液标准物质经重量-容量法配制后,进行均匀性和稳定性实验,浓度赋值后进行不确定度评定,诱惑红溶液的量值为100 mg/L,扩展相对不确定度为1.0%(k=2)。该溶液标准物质已批准为国家级标准物质,可为相关部门提供检测标准。     

Determination of methylmercury in marine sediment samples: Method validation and occurrence data

The determination of methylmercury (MeHg) in sediment samples is a difficult task due to the extremely low MeHg/THg (total mercury) ratio and species interconversion. Here, we present the method validation of a cost-effective fit-for-purpose analytical procedure for the measurement of MeHg in sediments, which is based on aqueous phase ethylation, followed by purge and trap and hyphenated gas chromatography–pyrolysis–atomic fluorescence spectrometry (GC–Py–AFS) separation and detection. Four different extraction techniques, namely acid and alkaline leaching followed by solvent extraction and evaporation, microwave-assisted extraction with 2-mercaptoethanol, and acid leaching, solvent extraction and back extraction into sodium thiosulfate, were examined regarding their potential to selectively extract MeHg from estuarine sediment IAEA-405 certified reference material (CRM). The procedure based on acid leaching with HNO₃/CuSO₄, solvent extraction and back extraction into Na₂S₂O₃ yielded the highest extraction recovery, i.e., 94 ± 3% and offered the possibility to perform the extraction of a large number of samples in a short time, by eliminating the evaporation step. The artifact formation of MeHg was evaluated by high performance liquid chromatography coupled to inductively coupled plasma mass spectrometry (HPLC–ICP–MS), using isotopically enriched Me²⁰¹Hg and ²⁰²Hg and it was found to be nonexistent. A full validation approach in line with ISO 17025 and Eurachem guidelines was followed. With this in mind, blanks, selectivity, working range (1–800 pg), linearity (0.9995), recovery (94–96%), repeatability (3%), intermediate precision (4%), limit of detection (0.45 pg) and limit of quantification (0.85 pg) were systematically assessed with CRM IAEA-405. The uncertainty budget was calculated and the major contribution to the combined uncertainty (16.24%, k = 2) was found to arise from the uncertainty associated with recovery (74.1%). Demonstration of traceability of measurement results is also presented. The validated measurement procedure was applied to the determination of MeHg incurred in sediments from a highly polluted and scarcely studied area in the Caribbean region.     

肌酐纯度标准物质的定值方法及其不确定度评定研究

通过定性及定量分析,研究了肌酐纯度标准物质的定值方法,并进行了定值分析的不确定度评定。首先使用三重四极杆质谱仪及核磁共振谱仪(氢谱)对肌酐样品进行定性分析,然后采用质量平衡法(包括液相色谱法、水分、灰分、挥发性物质和无机元素分析)与定量核磁共振法共同对肌酐纯度标准物质进行准确定值,最后对定值结果进行不确定度评定。肌酐的定值结果为99.7%,扩展不确定度为0.4%。该研究对于实际检测中肌酐的准确测定及临床上相关疾病的正确诊断治疗具有重要意义,且经过定值的肌酐纯品还可做定量核磁共振法的定量内标使用。定量分析后的肌酐经过均匀性检验和稳定性考察后可申报为国家标准物质。     

Gravimetric analysis of high purity tellurium for purity evaluation

Gravimetric analysis was utilized for purity evaluation of high purity tellurium, which was a raw material of a tellurium standard solution. Using a method in which tellurium was precipitated as elemental tellurium with hydrazinium after dissolution of the high purity tellurium, an avoidable positive error (ca. 0.02%) was observed. It seems that a cause of the positive error is oxidation of the tellurium or occlusion of the solvent during the precipitation. On the other hand, when the precipitated elemental tellurium was further converted into tellurium oxide by heating at 600 °C, the positive error disappeared. Finally, the purity of the starting tellurium was evaluated from the recovery of tellurium oxide with sufficient accuracy for a raw material of a tellurium standard solution (0.017% expanded uncertainty (k = 2)).     

Separation of Tic-hydantoin enantiomers,potent sigma-1 agonists,by high performance liquid chromatography and capillary electrophoresis

Stereospecific separations of seven Tic-hydantoin sigma-1 agonists were performed by both HPLC method using derivatized cellulose and amylose chiral stationary phases and capillary electrophoresis (CE) method using neutral and anionic cyclodextrins added in the background electrolyte (BGE). An optimal baseline separation (R_s > 3.3 with analysis times < 25 min) was readily obtained with all silica-based celluloses and amyloses using a normal-phase methodology. CE was used as an alternative technique to HPLC for the Tic-hydantoin derivatives separation. The enantiomers were fully resolved with highly sulfated β-cyclodextrins at pH 2.5 (R_s > 1.5 with analysis times <11 min). Both methods were validated in terms of linearity, detection and quantification limits. They were used to check the enantiomeric purity of the enantiomers.     

Uncertainty of measurement for summed masses: application to controlled substances

Because sentences for drug possession depend on the mass of the seized drug, testing laboratories must often determine the summed mass of numerous items submitted under a single case. One common practice for this purpose is to continue analyzing and weighing samples until a legal threshold is passed, at which point it is important to inform the court whether the summed mass is significantly above the threshold, or only marginally so. This paper develops a means for estimating the uncertainty of the summed mass for the common situation where the readability, d, of the balance dominates the uncertainty. It is seen that for all sample sizes the uncertainty, UM, is given by the remarkable simple expression UM = (d/2) × [N + SQRT(3N)] + N × CCE, where N is the number of items and CCE is the absolute value of the calibration check error. In most instances, this can be further simplified to UM = N × d.     

Certified calibration solution reference material for the determination of perfluorooctane sulfonate from the National Metrology Institute of Japan (NMIJ)

A new calibration solution reference material for the determination of perfluorooctane sulfonate anion (PFOS) and its salts has been issued as a certified reference material (CRM) by the National Metrology Institute of Japan, National Institute of Advanced Industrial Science and Technology (NMIJ/AIST). The purity amount-of-substance fraction of raw material potassium perfluorooctane sulfonate (K-PFOS) was evaluated based on the results obtained using the freezing point depression method, and the purity mass fraction of the raw material was calculated using the average molar mass of impurities, the molar mass of K-PFOS, and the purity amount-of-substance fraction. The certified concentration of this CRM was obtained by multiplying the dilution ratio of the raw material in a prepared solution (methanol) determined from the gravimetric blending method by the purity of the raw material. The preparation concentration of K-PFOS as a certified value of NMIJ CRM 4220-a was determined to be 9.93?mg?kg^?1. In addition, the standard uncertainty of the certified value was evaluated from the purity evaluation as well as from sample inhomogeneity, instability, and preparation variation obtained from LC/MS measurements of different gravimetrically prepared solutions of the NMIJ CRM. Consequently, the expanded uncertainty was estimated to be 0.15?mg?kg^?1 with a coverage factor k?=?2 corresponding to the half-width of estimated confidence interval of approximately 95%.     

Efficient isolation of the subunits of recombinant and pituitary glycoprotein hormones

Complete dissociation into subunits was attained by incubating Chinese hamster ovary (CHO)-derived or native human thyrotropin, follitropin and lutropin overnight at 37 °C in acetic acid. The α-and β-subunits of the pituitary glycoprotein hormones were rapidly and quantitatively isolated by reversed-phase high-performance liquid chromatography (RP-HPLC). A dissociation efficiency of >98% was obtained on the basis of mass determinations of the heterodimers and subunits carried out via mass spectrometry. CHO-derived or native subunits were isolated on a C₄ column (80–90% total recovery) and characterized comparatively for purity, hydrophobicity, molecular mass and charge distribution by HPLC, mass spectrometry, sodium dodecylsulfate-polyacrylamide gel electrophoresis and isoelectric focusing. Thyrotropin was used as a model for showing that, after subunit reassociation, the in vivo bioactivity of the hormone was completely restored. The method described is mild, practical, flexible, and can be adapted to dissociate microgram amounts of native or recombinant glycoprotein hormones, allowing characterization of each subunit.     

诺龙甲醇溶液标准物质的研制

利用色-质联用法和红外光谱法对诺龙纯品原料定性后,采用高效液相色谱多家定值方法对诺龙纯度进行定值,经重量法配制,制得了诺龙甲醇溶液标准物质,并对制备的溶液标准物质进行均匀性和稳定性检验.对定值不确定度进行了评定,当诺龙甲醇溶液标准物质的浓度为1.00 mg/mL时,相对扩展不确定度为1.0%,k=2.     

Matrix solid phase dispersion-assisted BCR sequential extraction method for metal partitioning in surface estuarine sediments

The BCR (the Community Bureau of Reference) of the European Union sequential extraction scheme for metal partitioning in estuarine sediments has been accelerated by using a matrix solid phase dispersion (MSPD) approach. The MSPD assisted BCR procedure consists of passing the extractants proposed by conventional BCR protocol (0.11 M acetic acid, 0.1 M hydroxylammonium chloride and 8.8 M hydrogen peroxide plus 1 M ammonium acetate) through the dispersed sample packaged inside a disposable syringe. Different silica-, magnesium- and aluminium-based materials were tested as dispersing agents and sea sand was found to offer the best performances. Variables for assisting the three stages of the BCR protocol were optimized, and accurate results were obtained when assisting the first and the third stages (exchangeable and oxidizable fractions, respectively). However, lack of accuracy was observed when assisting the second step (reducible fraction) and this result agrees with most of the assisted BCR procedures for which extracting the reducible fraction is the most troublesome stage. The organic matter oxidation (third stage) was successfully assisted by passing hydrogen peroxide at 50 °C through the dispersed sample inside de syringe just before passing ammonium acetate. Therefore, the time-consuming and unsafe conventional organic matter oxidation processes, commonly performed even for microwave/ultrasounds assisted BCR procedures, are totally avoided. Inductively coupled plasma-mass spectrometry (ICP-MS) was used as a selective detector. The target elements were Cd, Co, Cr, Mn, Ni, Sr and Zn (first stage), Cd, Co and Ni (second stage), and Co, Cr, Mn, Ni, Sr and Zn (third stage). Repeatability of the method (n = 7) was good, and RSDs values of 9, 10, 10, 8, 8, 3 and 8% was obtained for Cd, Co, Cr, Mn, Ni, Sr and Zn, respectively (first stage); 10, 9 and 9% for Cd, Co and Ni, respectively (second stage); and 6, 2, 3, 4, 7 and 9% Co, Cr, Mn, Ni, Sr and Zn, respectively (third stage). The procedure was also validated by analysing two certified reference materials (CRM 601 and CRM 701). Good accuracy was obtained for the target elements extracted at the first stage: Cd (4.0 ± 0.1 and 7.3 ± 0.09 μg g⁻¹ in CRM 601 and CRM 701, respectively), Cr (0.36 ± 0.008 and 2.21 ± 0.08 μg g⁻¹ in CRM 601 and CRM 701, respectively), Ni (8.0 ± 0.3 and 15.4 ± 0.3 μg g⁻¹ in CRM 601 and CRM 701, respectively) and Zn (262 ± 3 and 203 ± 3 μg g⁻¹ in CRM 601 and CRM 701, respectively). Also, good accuracy was observed for elements extracted at the third step: Cd (1.8 ± 0.09 and 0.29 ± 0.03 μg g⁻¹ in CRM 601 and CRM 701, respectively), Cr (145 ± 4 μg g⁻¹ in CRM 701), Ni (8.2 ± 0.7 and 15.1 ± 0.5 μg g⁻¹ in CRM 601 and CRM 701, respectively) and Zn (45 ± 0.7 μg g⁻¹ in CRM 701).     

Preparative isolation and purification of isobenzofuranone derivatives and saponins from seeds of Nigella glandulifera Freyn by high-speed counter-current chromatography combined with gel filtration

Although the medicinal plant and food Nigella glandulifera Freyn has been researched for decades, isobenzofuranones have never been isolated before. Two isobenzofuranone derivatives and two saponins were successfully separated and purified from seeds of N. glandulifera Freyn by high-speed counter-current chromatography (HSCCC) with the optimized two-phase solvent system, n-hexane-ethyl acetate–methanol–water (7:3:5:5, v/v). Salfredin B₁₁ (22.1 mg, HPLC purity 95.3%), 5, 7-dihydroxy-6-(3-methybut-2-enyl) isobenzofuran-1(3H)-one (18.9 mg, HPLC purity 97.3%) and crude sample 2 (555 mg) were separated from 600 mg of ethyl acetate extract of N. glandulifera Freyn. Following a cleaning-up step by chromatography on Sephadex LH-20, hederagenin (12 mg) and 3-O-[β-d-xylopyranosyl-(1 → 3)-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl]-hederagenin (45 mg) were separated from sample 2. All of the fractions before peak II were collected and subjected to a Sephadex LH-20 column and eluted by methanol, two of triterpene saponins (12 mg of hederagenin and 45 mg of 3-O-[β-d-xylopyranosyl-(1 → 3)-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl]-hederagenin) were isolated. The structures of peak fractions were identified by IR, electron ionization MS, ¹H NMR and ¹³C NMR. 5, 7-Dihydroxy-6-(3-methybut-2-enyl) isobenzofuran-1(3H)-one was isolated for the first time from higher plant and salfredin B11 was isolated for the first time in this plant.