A comparative uncertainty study of the calibration of macrolide antibiotic reference standards using quantitative nuclear magnetic resonance and mass balance methods

This study introduces the general method of quantitative nuclear magnetic resonance (qNMR) for the calibration of reference standards of macrolide antibiotics. Several qNMR experimental conditions were optimized including delay, which is an important parameter of quantification. Three kinds of macrolide antibiotics were used to validate the accuracy of the qNMR method by comparison with the results obtained by the high performance liquid chromatography (HPLC) method. The purities of five common reference standards of macrolide antibiotics were measured by the ¹H qNMR method and the mass balance method, respectively. The analysis results of the two methods were compared. The qNMR is quick and simple to use. In a new medicine research and development process, qNMR provides a new and reliable method for purity analysis of the reference standard.     

High performance liquid chromatography - Quantitative nuclear magnetic resonance - High performance liquid chromatography for purity measurement of human insulin

Quantitative Nuclear Magnetic Resonance (qNMR) is a reliable quantitative spectroscopic technique, wherein the intensity of a resonance line is directly proportional to the number of resonant nucleus, and the absolute content of the compound can be determined, this means the inorganic stabilizer in the sample would not affect the result of qNMR. High performance liquid chromatography (HPLC) is a common analytical method with a high separation capacity. This study combined HPLC and qNMR, to measure the purity of Human Insulin (HI). It started from an original HI. The first step is purifying the original HI by HPLC to get a purified HI, with organic purity of 99.78%. The second step is assessing the absolute content of the purified HI by qNMR, and got 40.25%. The third step is measuring the purity of original HI by HPLC again, using the purified HI as the reference material. This method, called HPLC-qNMR-HPLC, is more accurate (84.12%?±?1.14%) than the traditional IDMS (isotope dilution mass spectrometry) method (86.6%?±?3.4%). This study expanded the application of qNMR to proteins with molecular weight of about 5800, and showed that this method can be widely used in measuring the purity of macromolecular proteins.     

Using high-performance quantitative NMR (HP-qNMR®) for certifying traceable and highly accurate purity values of organic reference materials with uncertainties <0.1 %

Quantitative nuclear magnetic resonance (qNMR) in combination with metrological weighing is optimised to demonstrate the power of the qNMR measurement method. It is shown that with ¹H-qNMR it is possible to certify the purity of organic reference materials (expressed as mass fraction) with relative expanded uncertainties of <0.1 % for a 95 % confidence interval (k = 2). Following well-defined selection criteria, a set of twelve different chemical compounds is evaluated and certified to serve as internal references for ¹H-qNMR measurements. A series of comparison measurements is made amongst a subset of the selected compounds. The purity of maleic acid is determined by six different ¹H-qNMR measurement series, and all results show full consistency. All the six mean values are covered within the range of ±0.05 %. In two more measurement series, four different nuclei are analysed within the same sample against one calibrator. Even with non-optimised signal intensity ratios and varying signal pattern, a high consistency was obtained. Therefore, the validity and robustness of ¹H-qNMR measurement results are demonstrated. ¹H-qNMR measurement results are directly traceable to a variety of internationally accepted primary reference materials, and therefore, traceability to SI units is obtained. All experiments are performed under ISO/IEC 17025 and ISO Guide 34 accreditation.     

Purification of folic acid candidate reference material by preparative high-performance liquid chromatography

An effective process for the purification of folic acid candidate reference material with preparative high-performance liquid chromatography (Pre-HPLC) was developed in this study. During the process of experimental operation, parameters including the influences of mobile phase, flow rate, and injection volume on the purity and yield were investigated, and the optimized conditions were as follows: the mobile phase was acetonitrile and water in a gradient mode with flow rate of 16?mL/min and injection volume of 2.0?mL at concentration of 10?mg/mL. Under the conditions, the purity and yield of folic acid product were up to 99.4% and 21.0%, respectively, whereas the purity of folic acid raw material was 95.2%. The purified folic acid product was characterized by LC–MS, HPLC, Karl Fischer coulometer, and quantitative nuclear magnetic resonance (qNMR). Results proved that the main component of the product was folic acid and the purities determined by HPLC and qNMR were consistent. Two impurities including N-(4-aminobenzoyl)-L-glutamic acid and pteroic acid were further quantified by LC–MS. Compared with the recrystallization approach, the purity of folic acid obtained by Pre-HPLC increased from 98.2% to 99.4%.     

The application of qNMR for the determination of rosuvastatin in tablet form

In this study, quantative nuclear magnetic resonance (qNMR) method was used to determine the content of rosuvastatin in tablet. Linearity, range, limit of detection (LOD), limit of quantification (LOQ), accuracy, and precision were determined in validation study of rosuvastatin. Furthermore, validation study of rosuvastatin was performed with high performance liquid chromatography (HPLC). Uncertainties of qNMR and HPLC methods were determined using per EURACHEM/CITAC Guide CG 4 (3th edition), quantifying uncertainty in analytical measurement. qNMR and HPLC methods were linear in the ranges of 0.10 - 5.00 mg/mL and 0.001 - 0.0995 mg/mL, respectively and these lineraties indicate very good linearity performance with regression coefficients (R2 value) above > 0.99. Moreover, LOD and LOQ values using qNMR method were observed as 0.25 mg/mL and 0.80 mg/mL, respectively. These values using HPLC method were found as 0.00051 µg/mL and 0.001695 µg/mL, respectively. The strengths and weaknesses of qNMR method and HPLC method were determined with spectral emphasis on the role of identical reference standards in qualitive and quantitative analyses. It was found that qNMR method is simple, efficient, reliable, and accurate method. Moreover, qNMR method is an easy, practical, and useful method for the validation and optimization of rosuvastatin in the tablet.     

Quantitative 1H Nuclear Magnetic Resonance Method for Assessing the Purity of Dipotassium Glycyrrhizinate

A simple, rapid, accurate, and selective quantitative method based on ¹H nuclear magnetic resonance (qNMR) was successfully established and developed for assessing the purity of dipotassium glycyrrhizinate (KG). In this study, using potassium hydrogen phthalate and fumaric acid as internal standard (IS), several important experimental parameters, such as relaxation delay and pulse angle, were explored. Reliability, specificity, linearity, limit of quantification, precision, stability, and accuracy were also validated. Calibration results obtained from qNMR were consistent with those obtained from HPLC coupled with ultraviolet detection. The proposed method, independent of the reference standard substance, is a useful, reliable, and practical protocol for the determination of KG and glycyrrhizin analogs.     

Uncertainty measurement for automated macro program-processed quantitative proton NMR spectra

The evaluation of a fully automated quantitative proton nuclear magnetic resonance spectroscopy (qNMR) processing program, including the determination of its processing uncertainty, and the calculations of the combined uncertainty of the qNMR result, is presented with details on the use of a trimmed purity average. Quantitative NMR spectra (1359) were collected over a 4-month period on various concentrations of pseudoephedrine HCl dissolved in D₂O (0.0610 to 93.60 mg/mL) containing maleic acid (the internal standard) to yield signal-to-noise ratios ranging from 3 to 72,000 for analyte integral regions. The resulting 5436 purities exhibited a normal distribution about the best estimate of the true value. The median absolute deviation (MAD) statistical method was used to obtain a model of uncertainty relative to the signal-to-noise of the analyte’s integral peaks. The model was then tested using different concentrations of known purity chloroquine diphosphate. qNMR results of numerous illicit heroin HCl samples were compared to those obtained by capillary electrophoresis. Graphical Abstract

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Quantification of multiple compounds containing heterogeneous elements in the mixture by one‐dimensional nuclear magnetic resonance spectroscopy of different nuclei using a single universal concentration reference

One‐dimensional (1D) quantitative NMR (qNMR) is a useful tool for concentration determination due to its experimental simplicity and the direct proportionality of the integrated signal area to the number of nuclei spin. For complex mixtures, however, signal overlapping often in one‐dimensional quantitative ¹H NMR (1D ¹H qNMR) spectrum limits the accurate quantification of individual compound. Here, we introduced employing joint 1D qNMR methods of different nuclei, such as ¹H and ³¹P (or/and ¹⁹F), to quantify multiple compounds in a complex mixture using a single universal concentration reference. When the concentration ratio of several compounds containing different elements in a complex mixture is of interest, the result calculated from measured intensities from 1D qNMR of different nuclei is independent of the gravimetric error from the reference. In this case, the common reference also serves as a ‘quantitative bridge’ among these 1D qNMR of different nuclei. Quantitative analysis of choline, phosphocholine, and glycerophosphocholine mixture is given as an example using trimethylphosphine oxide ((CH₃)₃P(O)) as concentration reference. Compounds containing multiple elements, such as tetramethylammonium hexafluorophosphate (N⁺(CH₃)₄PF₆^?), are proposed as the common concentration reference for ¹H, ¹³C, ¹⁵N, ³¹P, and ¹⁹F qNMR for the quantitative analysis of complex mixture containing these different elements. We anticipate that the proposed joint 1D qNMR approach using a universal concentration reference will be a valuable alternative for simultaneous quantification of multiple compounds in a complex mixture due to its accuracy and single and simple sample preparation. Copyright © 2014 John Wiley & Sons, Ltd.     

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。     

定量核磁法测定CL-20标准物质中有机杂质的含量

采用定量核磁共振波谱法(qNMR)测定六硝基六氮杂异伍兹烷(CL-20)标准物质中有机杂质的含量。核磁谱图解析证明,主要有机杂质成分为残余溶剂乙酸乙酯和中间体五硝基-乙酰基六氮杂异伍兹烷(MPIW),以不含四甲基硅烷(TMS)的氘代丙酮为溶剂,将六甲基二硅醚的四氯化碳标准溶液加入待测液中作为内标,以其谱峰(δ=0.06)作为内标峰对两种有机杂质进行定量分析。考察了延迟时间和采样次数对准确定量的影响,结果显示为确保定量结果的准确性,延迟时间D_1应不小于20 s,采样次数为32次。采用优化后的实验参数进行纯度分析,测得CL-20标准物质中有机杂质乙酸乙酯和MPIW的质量分数分别为0.03516%和0.156 2%,相对标准偏差(RSD)分别为0.91%和0.86%。     

Differential scanning calorimetry to measure the purity of polycyclic aromatic hydrocarbons

The application of differential scanning calorimetry (DSC) for purity determination is well documented in literature and is used amongst others in the analysis of pure organic crystalline compounds. The aim of this work is to examine whether the DSC method for purity determination consistently produces values for the purity of polycyclic aromatic hydrocarbons (PAHs) which are sufficiently accurate as required for the certification of reference materials. For this purpose, 34 different existing PAH certified reference materials were tested. The DSC results are shown to be consistent with the results obtained by other methods assessing the organic impurities content in PAHs, like gas chromatography (GC), high performance liquid chromatography (HPLC) and mass spectrometry. Significant differences between the measured values and the certified purity values were observed only in a limited number of cases.     

Purity determination of a new antifungal drug candidate using quantitative 1H NMR spectroscopy: Method validation and comparison of calibration approaches

Quantitative nuclear magnetic resonance (qNMR) is an analytical technique that offers numerous advantages in pharmaceutical applications including minimum sample preparation and rapid data collection times with no need for response factor corrections, being a powerful tool for assaying drug content in both drug discovery and early drug development. In the present work, we have applied qNMR, using both the internal standard and the electronic reference to access in vivo concentrations 2 calibration methods, to assess the purity of RI76, a novel antifungal drug candidate. NMR acquisition and processing parameters were optimized in order to obtain spectra with intense, well-resolved signals of completely relaxed nuclei. The analytical method was validated following current guidelines, demonstrating selectivity, linearity, accuracy, precision, and robustness. The calibration approaches were statistically compared, and no significant difference was observed when comparing the obtained results and their dispersion in terms of relative standard deviation. The proposed qNMR method may, therefore, be used for both qualitative and quantitative assessments of RI76 in early drug development and for characterization of this compound.     

固相萃取-定量核磁共振波谱法测定板蓝根饮片中的表告依春

建立了固相萃取(SPE)-定量核磁共振波谱(qNMR)技术测定板蓝根饮片中有效成分表告依春含量的方法.样品用水超声提取两次,采用SPE对提取液进行富集浓缩,用qNMR测定表告依春的含量.考察了样品预处理和qNMR实验条件对测定结果的影响,选择氘代二甲基亚砜为溶剂,用基准试剂邻苯二甲酸氢钾标定的2,3,5-三碘苯甲酸为内标,选择脉冲宽度P1=14.1 μs,延迟时间d1=5 s,扫描次数NS=256为qNMR定量测定表告依春的最佳实验条件.表告依春的定量峰为δ 5.365~5.399 (H-7b, d,1H).结果表明,日内测量精密度(RSD)为0.5%,日间精密度为0.8%,表告依春与三碘苯甲酸峰面积比与质量比的零截距标准曲线线性相关系数为0.9991,且斜率与理论值相符.根据响应值标准偏差和标准曲线斜率法确定此法测定表告依春的检测限(LOD)为0.05 mg/g;定量限(LOQ, S/N ≥ 150)为0.19 mg/g.包括样品提取过程的表告依春的回收率为97.4%~101.7%.采用本方法测定板蓝根饮片中的表告依春的含量为<0.19~1.26 mg/g.研究结果表明,采用SPE进行富集,扩大了qNMR的应用范围,可用于低含量复杂样品的定量分析.     

Importance of the definition of catalytic properties for the commutability of an enzyme reference material: example of lipase

Assays for pancreatic lipase activity in serum need the use of calibrators, but there is a lack of a reference method and of a reference material. Under routine conditions, comparability of lipase catalytic activities was found to be poor when lipase was determined in 50 patients’ specimens by a turbidimetric (Boehringer) and a colorimetric (Sigma) assay. Mean values of the results differed by a ratio of 2.39. Catalytic properties of three materials, two commercial calibrators and a home-purified preparation of human pancreatic lipase (HPL), have been compared in titrimetry at constant pH. Optimal common conditions were defined for the titration of lipase activity in the three materials. When using these titres for each calibrator, comparability was greatly improved (ratio = 1.25). This result indicates that a significant part of between-method discrepancy was due to the lack of a reference method for the titration of lipase calibrators. The inter-method behavior of each material was compared to that of patients’ specimens. Using HPL as calibrator, the comparability of patients’ results was still dramatically improved (ratio = 1.01). This study shows the importance of verifying the similarity of catalytic properties of an enzyme reference material to those of the corresponding enzyme in human specimens, in order to ensure its commutability. The use of validated commutable calibrators, traceable to certified reference materials, is an opportunity to improve the inter-method comparability of results in clinical enzymology.     

Reference materials for energy and temperature calibrations of DTA and DSC instrumentation in the range 100–300 K

Fourteen organic compounds, 1-pentene, 2-methylbutane, 2-methyl-1,3-butadiene (isoprene),n-pentane, 2-chloropropane, ethyl alcohol, 2,2,4-trimethylpentane,n-hexane,n-hepatane, 1,3-difluorobenzene, 1,2-dichloroethane, hexafluorobenzene, cyclohexane and diphenyl ether, as new reference materials for energy and temperature calibrations of differential thermal analysers (DTA) and differential scanning calorimeters (DSC) in the temperature range 100–300 K, were investigated. The purities, triple point and transition temperatures, fusion and transition enthalpies of these compounds were measured using a low-temperature DTA apparatus developed by the CTM of the CNRS in Marseille. The results obtained were compared with those produced by adiabatic precision calorimetry. Our data are in good agreement with literature values. Enthalpies of fusion and transition, triple point and transition temperatures are reproduced within ±1% and ±0.1K, respectively. during the period 1991–2.     

Quantitative 1H NMR spectroscopy (qNMR) in the early process development of a new quorum sensing inhibitor

2-methyl-5,6,7,8-tetrahydro-2H-chromen-4(3H)-one (called 6-oxo) is presented as a new AI-1 quorum sensing inhibitor for Vibrio harveyi. The development of a chemical process to afford traceable materials for new biological assays demands the development of analytical methods to ensure their purity and quality. This work describes the use of quantitative ¹H nuclear magnetic resonance (NMR) spectroscopy (qNMR) to assess the purity of a sample of 6-oxo (99.88%) and a sample of its major process impurity (E)-1-(2-hydroxycyclohex-2-en-1-yl)but-2-en-1-one (called HCB; 98.28%). To explore the scope of the use of qNMR to quantify the amount of low-content components in samples related to the chemical process for 6-oxo synthesis, this work also determined the amount of 6-oxo in two HCB samples: (a) the high-purity HCB sample described above and (b) a crude HCB sample collected during the chemical process. Despite the complexity of the crude sample, the amount of 6-oxo was readily assessed and could help to estimate the extent to which 6-oxo was already formed during the HCB synthesis. This information can help the understanding of how the process parameters can be modified to improve the performance of the whole process, by controlling the reaction mechanisms working at each step of this chemical process. In this context, our results reinforce qNMR as a complementary analytical tool for the quantification of the main component found in a sample, contributing to the standardization of reference materials and thus allowing the development of analytical methods for process control and traceability of the samples used for biological assays.     

An integrated approach of homogenization-ultrasound-assisted extraction followed by high-speed countercurrent chromatography for rapidly preparative separation naphthoquinones and diarylheptanes from Juglands mandshurica Maxim. exocarp

This research investigated the effectiveness of an integrated method for the extraction and separation of naphthoquinones and diarylheptanes from exocarp of Juglands mandshurica Maxim. (namely, green walnut husks). The target compounds were obtained by ultra-turrax homogenization (UTH) coupled with ultrasound-assisted extraction (UAE) technology followed by high-speed countercurrent chromatography (HSCCC). The UTH-UAE extraction method achieved higher efficiency with 2.49- and 2.36-fold to those by UAE, and 1.39- and 1.34-fold to those by UTH in a short time. HSCCC was adopted for further separation and purification; six target compounds, namely, regiolone (RE), juglone (JU), myricatomento-genin (MG), galleon (GA), 2-oxatrycyclo[13.2.2.13,7]eicosa-3,5,7(20),15,17,18-hexaen-10-16-diol (OE), and juglanin A (JA), were separated with more than 95.37% purities and more than 84.71% final recovery rates, respectively. In this study, the integrated strategy of extraction and separation could get high purity compounds quickly, which would provide time and solvent saved method for the natural products separation from plants.     

Determining and reporting purity of organic molecules: why qNMR

Although NMR has been routinely used to determine/estimate relative number of protons for structure elucidation, it has been rarely used to determine and report the purity of organic compounds. Through this paper, we want to emphasize on routine use of quantitative NMR (qNMR) for this purpose. The results of qNMR can be routinely considered as documentation of purity much like other established methods (HPLC, elemental analysis and differential scanning calorimetry). qNMR is a fast, easy, accurate and non‐destructive alternate to speed up the whole analytical process and serves the purpose of both identification and purity determination of compounds using single technique. Copyright © 2012 John Wiley & Sons, Ltd.     

Evaluation of certified reference materials for oxidation-reduction titration by precise coulometric titration and volumetric analysis

The accuracy and uncertainty of coulometric titration of Japanese certified reference materials (CRMs) for oxidation-reduction titration were examined in this study. The results for potassium dichromate, sodium oxalate, and potassium iodate are presented. Potassium dichromate was directly determined by coulometric titration, and sodium oxalate and potassium iodate were determined by volumetric analysis using potassium dichromate assigned by coulometry.The uncertainty of the method was investigated by examining the dependency on the sample size and on the electrolysis current. Changes in the titration parameters did not result in any significant effects on the titration results. The coulometric system used primarily consists of a constant current source, timer, switching circuit, indicator unit, and voltmeter. They were controlled using the coulometry software by a PC/AT compatible computer. A highly automated coulometric system achieves repeatabilities of less than one part in 30,000 (k = 2) and uncertainties of less than one part in 15,000 (k = 2). In addition, using volumetric method, SI units traceable sodium oxalate and potassium iodate (purity standards for redox reaction) CRMs were developed.Reference materials for volumetric analysis are the most basic substances used in analytical chemistry. These materials are analyzed by several analytical methods and are produced globally; however, their purities have not been compared at the international level. Therefore, the relationship between the purity and reliability of these materials has not yet been established. In this paper, we determine the relationship between these parameters by titrating each material obtained from different laboratories.     

Quality evaluation and species differentiation of Rhizoma coptidis by using proton nuclear magnetic resonance spectroscopy

Rhizoma coptidis, a broadly used traditional Chinese medicine, derives from the dried rhizomes of Coptis chinensis Franch, Coptis deltoidea C.Y. Cheng et Hsiao and Coptis teeta Wall. Quantitative determination of protoberberine alkaloids in R. coptidis is critical for controlling its quality. In this study, a rapid, simple and accurate quantitative ¹H NMR (qNMR) method was developed for simultaneous determination of berberine, jatrorrhizine, epiberberine, coptisine, palmatine and columbamine in R. coptidis from the three species. Method validation was performed in terms of selectivity, precision, repeatability, stability, accuracy, robustness and linearity. The average recoveries obtained were in the range of 96.9–102.4% for all the six alkaloids. In addition, the qNMR data were analyzed with analysis of variance (ANOVA), hierarchical clustering analysis (HCA) and principal component analysis (PCA), and the results showed that the contents of the active alkaloids have significant difference among the three species. Compared with the conventional HPLC approach, the proposed qNMR method was demonstrated to be a powerful tool for quantifying the six alkaloids due to its unique advantages of high robustness, rapid analysis time and no need of standard compounds for calibration curves preparation. These findings indicate that this method has potential as a reliable method for quality evaluation of herb medicines, especially for protoberberine alkaloid-containing ones.