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.     

A new traceability scheme for the development of international system-traceable persistent organic pollutant reference materials by quantitative nuclear magnetic resonance

Quantitative nuclear magnetic resonance (qNMR) was used for the purity determination of neat compounds of persistent organic pollutants (POPs). qNMR is a unique quantitative method that is not only traceable to the International System of Units (SI), but it also does not require a standard of its own. The purities of the POP compounds determined in this work were traceable to a single certified reference material (CRM), which is extremely attractive for reference material producers. The purities observed by qNMR were equivalent to those observed by gas chromatography with flame ionization detection (GC/FID) or a differential scanning calorimetry (DSC) combined with a thermogravimetric analyzer (TGA). The uncertainties obtained by the qNMR method were comparable to being slightly larger than those observed by DSC.     

Method development and validation: quantitation of telmisartan bulk drug and its tablet formulation by 1H NMR spectroscopy

The quantitative NMR (qNMR) spectroscopy is nowadays a new tool for the determination of pharmaceutical potent biologically active molecules in bulk drug and its tablet formulation than the other analytical techniques. Herein, qNMR method was developed for an anti‐hypertensive drug, telmisartan in bulk drug and its tablet formulation. The precise method was developed by using malononitrile as an internal standard. The methylene signal of telmisartan appeared at δ = 5.46 ppm (singlet) relative to the signal of malononitrile at δ = 3.59 ppm (singlet) in CDCl₃, as an NMR solvent. The development and validation of the method were carried out as per International Conference on Harmonization guidelines. The method was found to be linear (r² = 0.9999) for 0.5 to 3.5 mg/ml in the drug concentration range. The relative standard deviation for accuracy and precession was not more than 2.0%. The sensitivity of the method was carried out by limit of detection and a limit of quantification, at 0.05 and 0.2 mg/ml, respectively, concentration. The robustness of the method was studied by changing parameters as well as different solvent manufacturer company. The result shows that method was accurately developed for quantification of telmisartan in pharmaceutical dosage form. The developed method by ¹H NMR spectroscopy is comparatively easy and more precise with respect to the other analytical tools. Copyright © 2016 John Wiley & Sons, Ltd.     

Quantitative determination and validation of avermectin B1a in commercial products using quantitative nuclear magnetic resonance spectroscopy

Nuclear magnetic resonance is defined as a quantitative spectroscopic tool that enables a precise determination of the number of substances in liquids as well as in solids. There is few report demonstrating the application of NMR in the quantification of avermectin B_1a (AVB_1a); here, a proton nuclear magnetic resonance spectroscopy (¹H NMR) using benzene [1‐methoxy‐4‐(2‐nitroethyl) (PMN)] as an internal standard and deuterochloroform as an NMR solvent was tested for the quantitative determination of AVB_1a. The integrated signal of AVB_1a at 5.56 ppm and the signal of PMN at 8.14 ppm in the ¹H NMR spectrum were used for quantification purposes. Parameters of specificity, linearity, accuracy, precision, intermediate precision, range, limit of detection (LOD), limit of quantification (LOQ), stability and robustness were validated. The established method was accurate and precise with good recovery (98.86%) and relative standard deviation (RSD) of assay (0.34%) within the linearity of the calibration curve ranging from 5.08 to 13.58 mg/ml (R² = 0.9999). The LOD and LOQ were 0.009 and 0.029 mg/ml, which indicated the excellent sensitivity of the method. The stability of the method was testified by a calculated RSD of 0.11%. The robustness was testified by modification of four different parameters, and the differences among each parameter were all less than 0.1%. Comparing with the assay described by the manufacturer of avermectin tablets, there was no significant difference between the assay obtained by HPLC and quantitative NMR (qNMR), which indicated qNMR was a simple and efficient method for the determination of AVB_1a in commercial formulation products. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

Quantitative NMR (qNMR) for pharmaceutical analysis: The pioneering work of George Hanna at the US FDA

In the last two decades, quantitative NMR (qNMR) has become increasingly important for the analysis of pharmaceuticals, chemicals, and natural products including dietary supplements. For the purpose of quality control and chemical standardization of a large variety of pharmaceutical, chemical, and medicinal products, qNMR has proven to be a valuable orthogonal quantification method and a compelling alternative to chromatographic techniques. This work reviews a fundamental component of the early development of qNMR, reflected in the pioneering work of the late George M. Hanna during the years between 1984 and 2006 at the US Food and Drug Administration (FDA). Because Hanna performed the majority of his groundbreaking work on a 90‐MHz instrument, his legacy output connects with recent progress in low‐field benchtop NMR instrumentation. Hanna gradually established the utility of qNMR for the routine quality control analyses practiced in pharmaceutical and related operations well ahead of his peers. His work has the potential to inspire new developments in qNMR applied to small molecules of biomedical importance.     

Sodium quantitation in soft drinks: A rapid methodology by qNMR

High sodium dietary intake has a positive association with an increase in blood pressure and can be correlated with risk factors of disease. Considering that the World Health Organization recommends a sodium intake lower than 2 g day^-1 for adults, the hidden sodium content in processed foods is an important factor that compromises the assessment of a healthy diet. Accordingly, the present paper aimed to quantitate the sodium content of conventional soft drinks and their diet versions using nuclear magnetic resonance spectroscopy as a quantitative analytical technique (²³Na qNMR). The results show one free sodium signal (Na⁺) displayed as a singlet at 0 ppm in the ²³Na NMR spectrum, making its quantitation highly specific. This signal alone was used to directly determine the concentration of sodium in soft drinks. Flame photometry analysis was used to validate the method, and an excellent linearity was found in qNMR analysis (r = 0.9994) in comparison with flame photometry (r = 0.9958). In addition, a good correlation was found between sodium concentrations obtained by ²³Na NMR and flame photometry in the evaluated commercial soft drinks. Since the intensity of the resonance line is directly proportional to the number of nuclei (spins), the concentration of sodium in soft drinks can be determined via this straightforward method without the need for external calibration. The experimental acquisitions are fast (approximately 15 min), allowing the analysis of several samples in a short period of time. This is a novel alternative for sodium quantitation using an efficient NMR methodology.     

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.     

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.     

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.     

Quantitative determination and validation of octreotide acetate using 1H‐NMR spectroscopy with internal standard method

Quantitative nuclear magnetic resonance (qNMR) is a well‐established technique in quantitative analysis. We presented a validated ¹H‐qNMR method for assay of octreotide acetate, a kind of cyclic octopeptide. Deuterium oxide was used to remove the undesired exchangeable peaks, which was referred to as proton exchange, in order to make the quantitative signals isolated in the crowded spectrum of the peptide and ensure precise quantitative analysis. Gemcitabine hydrochloride was chosen as the suitable internal standard. Experimental conditions, including relaxation delay time, the numbers of scans, and pulse angle, were optimized first. Then method validation was carried out in terms of selectivity, stability, linearity, precision, and robustness. The assay result was compared with that by means of high performance liquid chromatography, which is provided by Chinese Pharmacopoeia. The statistical F test, Student's t test, and nonparametric test at 95% confidence level indicate that there was no significant difference between these two methods. qNMR is a simple and accurate quantitative tool with no need for specific corresponding reference standards. It has the potential of the quantitative analysis of other peptide drugs and standardization of the corresponding reference standards.     

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.     

Optimal flip angle for robust practical quantitative NMR measurement using a fixed pulse length

NMR quantification has been traditionally performed by using internal standards. Although methods using external reference in NMR quantification have been developed, the major obstacles in using external referencing method are the measurement deviations associated with changing sample conditions and the requirement of pulse width calibration for every sample in order to compensate these errors. The calibration process is time consuming and in some cases impossible. We developed a quantitative NMR method fixed pulse length (FIXPUL) for all measurements without sample-by-sample calibration. The method is based on the use of an optimal flip angle calibrated for an external standard so that the quantitative errors associated with the pulse width variations are minimized. FIXPUL can be implemented on most basic NMR spectrometers and is robust and easily automated. The method is applicable to a wide range of solution NMR samples in chemistry, biology, and drug research and discovery.     

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.     

Systematic development and validation of stability indicating micellar electrokinetic chromatography methods for early stage drug candidates

Suitable methods for assessment of purity, potency and stability of new drug substances and drug products are required to be rapidly developed and validated to provide appropriate data for early project development decisions. In order to routinely provide methods of consistent and suitable quality to meet increasingly aggressive timelines, systematic approaches to both develop and validate analytical technologies have been developed. Systematic approaches to evaluate separation parameters such as buffer pH, buffer ionic strength, surfactant concentration, organic modifiers, organic modifier concentration, applied voltage and temperature were evaluated for an early stage drug candidate. Techniques to improve method precision and ruggedness were also examined. Finally, the validation results from the micellar electrokinetic chromatography method utilizing an internal standard were compared against the simultaneously developed high-performance liquid chromatography method.     

Quantitative NMR for bioanalysis and metabolomics

Over the last several decades, significant technical and experimental advances have made quantitative nuclear magnetic resonance (qNMR) a valuable analytical tool for quantitative measurements on a wide variety of samples. In particular, qNMR has emerged as an important method for metabolomics studies where it is used for interrogation of large sets of biological samples and the resulting spectra are treated with multivariate statistical analysis methods. In this review, recent developments in instrumentation and pulse sequences will be discussed as well as the practical considerations necessary for acquisition of quantitative NMR experiments with an emphasis on their use for bioanalysis. Recent examples of the application of qNMR for metabolomics/metabonomics studies, the characterization of biologicals such as heparin, antibodies, and vaccines, and the analysis of botanical natural products will be presented and the future directions of qNMR discussed.     

Direct simultaneous analysis of plasma samples for a drug discovery compound and its hydroxyl metabolite using mixed-function column liquid chromatography-tandem mass spectrometry

A polymer-coated mixed-function (PCMF) column was evaluated for direct plasma injection for the simultaneous determination of a drug candidate and its hydroxyl metabolite by high-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS-MS) in support of pharmacokinetic studies. Each diluted monkey plasma sample containing internal standard was directly injected on to the PCMF column for sample clean-up, enrichment and chromatographic separation. The proteins and macromolecules were first eluted from the column while the drug molecules were retained on the bonded hydrophobic phase. The analytes retained on the column were then eluted with a strong mobile phase using a gradient separation technique at a constant flow rate of 1.0 ml min(-1). When not diverted, the column effluent was connected either to the atmospheric pressure chemical ionization (APCI) source or the electrospray ionization (ESI) source as part of the mass spectrometer system used for quantification. The calibration curve was linear over the range 5-2500 ng ml(-1) for both analytes. The retention times for the analytes and the internal standard were both consistent and no column deterioration was observed for at least 500 injections. The recovery through the column and reproducibility of the dosed compound and its hydroxyl metabolite in monkey plasma samples were > 90% (RSD < 6%). The total analysis time was < 8 min per sample. The analytical results obtained by the proposed direct plasma injection method were in good agreement with those obtained by the conventional LC-MS-MS method.     

Electrochemical valveless flow microsystems for ultra fast and accurate analysis of total isoflavones with integrated calibration

A novel strategy integrating methodological calibration and analysis on board on a planar first-generation microfluidics system for the determination of total isoflavones in soy samples is proposed. The analytical strategy is conceptually proposed and successfully demonstrated on the basis of (i) the microchip design (with the possibility to use both reservoirs), (ii) the analytical characteristics of the developed method (statically zero intercept and excellent robustness between calibration slopes, RSDs < 5%), (iii) the irreversible electrochemical behaviour of isoflavone oxidation (no significant electrode fouling effect was observed between calibration and analysis runs) and (iv) the inherent versatility of the electrochemical end-channel configurations (possibility of use different pumping and detection media). Repeatability obtained in both standard (calibration) and real soy samples (analysis) with values of RSD less than 1% for the migration times indicated the stability of electroosmotic flow (EOF) during both integrated operations. The accuracy (an error of less than 6%) is demonstrated for the first time in these microsystems using a documented secondary standard from the Drug Master File (SW/1211/03) as reference material. Ultra fast calibration and analysis of total isoflavones in soy samples was integrated successfully employing 60 s each; enhancing notably the analytical performance of these microdevices with an important decrease in overall analysis times (less than 120 s) and with an increase in accuracy by a factor of 3.     

Developing multiple high-throughput GLP methods for an investigational drug candidate in various matrices within a 96-well plate

In early pharmaceutical product development, an investigational drug candidate is typically dosed to various species for toxicological and pharmacokinetic studies. Most of these studies require multiple analytical methods that have to be validated with good laboratory practice (GLP) prior to the application in regulated studies. Usually, these analytical methods are developed in either a serial or parallel approach. For either approach, the development of multiple analytical methods takes tremendous work from scientists and instruments, and thus is not cost-effective. In this respect, a new strategy has been developed for simultaneous GLP method development using liquid chromatographic separation and tandem mass spectrometric detection. This high-throughput approach allows system suitability, carryover, calibration curve, accuracy, precision, matrix effect and selectivity to be evaluated in one 96-well plate. The strategy has been successfully implemented for multiple investigational drug candidates at Abbott Laboratories. The methods developed with this strategy are accurate, precise, selective, robust and matrix-independent. As an example, ABT-279 was used to demonstrate the feasibility of this strategy.     

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%。