The application of quantitative NMR for the facile,rapid and reliable determination of clindamycin phosphate in a conventional tablet formulation

Spectroscopic tools such as NMR can be applied to the quantitative analysis of active pharmaceutical ingredients with relative ease and accuracy. Here, we demonstrate the quantification of clindamycin phosphate (CLP) in a conventional tablet formulation, performed using potassium hydrogen phthalate (KHP) as the internal standard and deuterium oxide (D₂O) as the NMR solvent. The methyl protons signal of CLP at 0.72 ppm (triplet) relative to the signal of KHP at 7.37–7.40 ppm (multiplet) was used for quantification purposes using ¹H NMR. This method was shown to be specific and linear (r = 0.9997) within the CLP concentration range from 7.2 to 23.1 mg per 0.5 ml of D₂O. The maximum relative standard deviation (RSD) of accuracy and precision was calculated at 0.39% and 0.64%, respectively. The limits of detection (LOD) and quantification were 0.04 and 0.11 mg/ml, respectively. The method was highly stable with a calculated RSD of 0.03%. The robustness of the method was demonstrated by changing four different parameters, and the difference among each parameter was ≤ 0.78%. The findings of this work were in good agreement with previously reported conventional HPLC‐based approaches, highlighting its applicability in the determination of other active pharmaceutical ingredients in conventional formulations for quality control purposes. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

Quantitative analysis of malic and citric acids in fruit juices using proton nuclear magnetic resonance spectroscopy

¹H NMR spectroscopy was applied to the quantitative determination of malic and citric acids in apple, apricot, pear, kiwi, orange, strawberry and pineapple juices. Aspartic acid was studied as a potential interference. The effect of the sample pH on the chemical shifts of signals from malic, citric and aspartic acids was examined and a value of 1.0 was selected to carry out the determination. Integration of NMR signals at 2.89-2.95 and 3.00-3.04 ppm were used for calculating the concentration of malic and citric acids, respectively. At this pH the integrated signals were not overlapped. Sodium 3-(trimethylsilyl)tetradeuteropropionate (TSP) was used as an internal reference. The obtained results applying NMR procedures to analyze the juices from different fruits were compared to those obtained using enzymatic methods and both were in close agreement. The intra- and inter-day repeatability was tested for apple juice (7.86 g l⁻¹ malic acid, 0.32 g l⁻¹ citric acid) and apricot juice (5.06 g l⁻¹ malic acid, 4.79 g l⁻¹ citric acid) obtaining coefficients of variation lower than 3.4% for intra-day measures (n = 10) and lower than 3.8% for inter-day measures (n = 20).     

Ethanol determination in frozen fruit pulps: an application of quantitative nuclear magnetic resonance

This study reports the chemical composition of five types of industrial frozen fruit pulps (acerola, cashew, grape, passion fruit and pineapple fruit pulps) and compares them with homemade pulps at two different stages of ripening. The fruit pulps were characterized by analyzing their metabolic profiles and determining their ethanol content using quantitative Nuclear Magnetic Resonance (qNMR). In addition, principal component analysis (PCA) was applied to extract more information from the NMR data. We detected ethanol in all industrial and homemade pulps; and acetic acid in cashew, grape and passion fruit industrial and homemade pulps. The ethanol content in some industrial pulps is above the level recommended by regulatory agencies and is near the levels of some post‐ripened homemade pulps. This study demonstrates that qNMR can be used to rapidly detect ethanol content in frozen fruit pulps and food derivatives. Copyright © 2015 John Wiley & Sons, Ltd.     

Quantitation of memantine hydrochloride bulk drug and its tablet formulation using proton nuclear magnetic resonance spectrometry

The use of quantitative nuclear magnetic resonance spectrometry for the determination of non‐UV active memantine hydrochloride with relative simplicity and precision has been demonstrated in this study. The method was developed on a 500 MHz NMR instrument and was applied to determination of the drug in a tablet formulation. The analysis was performed by taking caffeine as an internal standard and D₂O as the NMR solvent. The signal of methyl protons of memantine hydrochloride appeared at 0.75 ppm (singlet) relative to the signal of caffeine (internal standard) at 3.13 ppm (singlet). The method was found to be linear (r² = 0.9989) in the drug concentration range of 0.025 to 0.80 mg/ml. The maximum relative standard deviation for accuracy and precision was <2. The limits of detection and quantification were 0.04 and 0.11 mg/ml, respectively. The robustness of the method was revealed by changing nine different parameters. The deviation for each parameter was also within the acceptable limits. The study highlighted possibility of direct determination of memantine hydrochloride in pure form and in its marketed tablet formulation by the use of quantitative NMR, without the need of derivatization, as is the requirement in HPLC studies. Copyright © 2016 John Wiley & Sons, Ltd.     

Quality assessment of Radix Codonopsis by quantitative nuclear magnetic resonance

Radix Codonopsis (Dangshen) is a famous traditional Chinese medicine and has long been used for replenishing energy deficiency, strengthening the immune system, lowering blood pressure and improving appetite in China, Japan and Korea. A highly specific quantification method using ¹H NMR has been developed for the simultaneous determination of novel quaternary ammonium alkaloids codotubulosine A and B, adenosine and 5-(hydroxymethyl)furfural in Radix Codonopsis materials Codonopsis pilosula, C. pilosula var. modesta, C. tangshen, C. tubulosa, C. subglobosa, C. clematidea, C. lanceolota and Campanumoea javanica collected from different regions of China and Taiwan. A solid-phase extraction with C-18 cartridge followed by elution with water can easily remove sugars the major components that may affect the determination of target constituents. In the ¹H NMR spectrum, the signals of N-CH₃ of codotubulosine A (δ 2.75) and B (δ 2.83), H-8 of adenosine (δ 8.15), and CHO signal of 5-(hydroxymethyl)furfural (δ 9.49) are well separated from other signals in [²H₄]methanol. The quantity of the compounds was calculated by the relative ratio of the integral values of the target peaks of each compound to the known amount of internal standard pyrazine. The described NMR method is found to be relatively simple, specific, precise and accurate for the quality control of Radix Codonopsis herbs and no reference compounds are required for calibration curves, in comparison to conventional HPLC methods, for instance.     

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.     

Spatial location of indomethacin associated with unimeric amphiphilic carrier macromolecules as determined by nuclear magnetic resonance spectroscopy

A combination of nuclear magnetic resonance (NMR) techniques including, proton NMR, relaxation analysis, two‐dimensional nuclear Overhauser effect spectroscopy, and diffusion‐ordered spectroscopy, has been used to demonstrate the spatial location of indomethacin within a unimolecular micelle. Understanding the location of drugs within carrier molecules using such NMR techniques can facilitate rational carrier design. In addition, this information provides insight to encapsulation efficiency of different drugs to determine the most efficient system for a particular bioactive. This study demonstrates that drugs loaded by the unimolecular amphiphile under investigation are not necessarily encapsulated but reside or localize to the periphery or interfacial region of the carrier molecule. The results have further implications as to the features of the unimolecular carrier that contribute to drug loading. In addition, evidence of drug retention associated with the unimolecular surfactant is possible in organic media, as well as in an aqueous environment. Such findings have implications for rational carrier design to correlate the carrier features to the drug of interest and indicate the strong retention capabilities of the unimolecular micelle for delivery applications. Copyright © 2016 John Wiley & Sons, Ltd.     

Conformational analysis of N-arachidonylethanolamide (anandamide) using nuclear magnetic resonance and theoretical calculations

The conformational properties of cis-5,8,11,14-eicosatetraenoylethanolamide (anandamide) were analysed by the combined use of NMR experimental results plus molecular simulations. The structure of anandamide was found to be a predominantly linear with a seven-atom ring of the ethanolamine group having a hydrogen bond which stabilizes the molecule. The vinylic group present has a cis conformation in solution. The terminal chain has a linear conformation and undergoes isotropic fast motion typical of this structure. Copyright © 2001 John Wiley & Sons, Ltd.     

Screening Brazilian commercial gasoline quality by hydrogen nuclear magnetic resonance spectroscopic fingerprintings and pattern-recognition multivariate chemometric analysis

The identification of gasoline adulteration by organic solvents is not an easy task, because compounds that constitute the solvents are already in gasoline composition. In this work, the combination of Hydrogen Nuclear Magnetic Resonance (¹H NMR) spectroscopic fingerprintings with pattern-recognition multivariate Soft Independent Modeling of Class Analogy (SIMCA) chemometric analysis provides an original and alternative approach to screening Brazilian commercial gasoline quality in a Monitoring Program for Quality Control of Automotive Fuels. SIMCA was performed on spectroscopic fingerprints to classify the quality of representative commercial gasoline samples selected by Hierarchical Cluster Analysis (HCA) and collected over a 6-month period from different gas stations in the São Paulo state, Brazil. Following optimized the ¹H NMR-SIMCA algorithm, it was possible to correctly classify 92.0% of commercial gasoline samples, which is considered acceptable. The chemometric method is recommended for routine applications in Quality-Control Monitoring Programs, since its measurements are fast and can be easily automated. Also, police laboratories could employ this method for rapid screening analysis to discourage adulteration practices.     

Nuclear magnetic resonance using electronic referencing: method validation and evaluation of the measurement uncertainties for the quantification of benzoic acid in orange juice

Quantitative nuclear magnetic resonance measurements have become more popular over the last decade. The introduction of new methods and experimental parameters has been of fundamental importance in the development of new applications. Amongst these new developments is the introduction of electronic referencing for quantifications. The use of electronic referencing eliminates errors in the analyses as a result of weighting of internal standards as well as undesired problems as a result of the solubility of the standards in the analyte solution and chemical interactions between the analyte and the internal standard. In this work, we have studied the quantification of a very important analyte in a food matrix, benzoic acid in orange juice, as a model to the validation and measurement uncertainty estimation of electronic referencing using ¹H NMR in food analyses. The referencing method applied was the pulse length‐based concentration measurement. Method was validated and showed good results for the precision and accuracy parameters evaluated. A certified reference material and a reference material candidate were analyzed, and extremely good results were obtained. Reported relative expanded uncertainties are in the 1.07–1.39% range that can be considered an extremely good performance for the analysis of a food complex matrix. Measurement uncertainty was evaluated by two different approaches, and the pulse calibrations for the samples and for the reference have been shown to account for approximately 80% of the total uncertainty of the measurement. Copyright © 2014 John Wiley & Sons, Ltd.     

Investigation of molecular motions of crosslinked polyepichlorohydrin by nuclear magnetic resonance spectroscopy

The molecular motion of crosslinked polyepichlorohydrin (PECH) is studied qualitatively by NMR techniques. The results of temperature dependence of ¹H T₂ and T₁ indicate that the crosslinking (crosslink density < 3%) restricts molecular motions of the polymer even far above its T_g. The ¹H T₁ minimum, corresponding to the large-scale chain-motion of crosslinked PECH, shifts to higher temperatures with increasing crosslink density. ¹H T₂ data also show that the crosslinking hinders free chain motions of the polymer above its T_g. The ¹³C T₁ relaxation time is sensitive to such motional changes as well. ¹³C linewidths of crosslinked PECHs vary with the crosslink density in both the swollen state and the solid state. The mechanism of ¹³C linewidth broadening of crosslinked polymers is discussed in detail. In the case of PECH, the linewidth broadening is caused by changing molecular environment due to crosslinking (such as presence of various chemical shift structures and freezing effects in conformational environment as chain mobility decreases), rather than increasing correlation times, which shorten the relaxation time (T₂) of polymer chains. © 1994 John Wiley & Sons, Inc.     

手性溶剂-NMR法测定2,4-滴丙酸对映体纯度的定量分析

研究了手性溶剂法测定2,4-滴丙酸的^1H、^13C谱,在满足NMR准确定量所要求的分离度和信噪比的条件下,能准确测定手性化合物的对映体纯度。比较了以对映体百分含量（R％）和对映体过量（ee）表示手性农药的对映体纯度的差别,发现以对映体百分含量代替对映体过量来表示手性农药的对映体纯度更为准确。     

Phosphate additives determination in meat products by 31-phosphorus nuclear magnetic resonance using new internal reference standard: hexamethylphosphoroamide

New ³¹P NMR internal reference standard - hexamethylphosphoroamide (HMPA) was applied for determination of added polyphosphates and their ionic forms in raw pork meat and meat products. Phosphate species were determined after extraction with a boric acid buffer (pH = 9) and EDTA solution, using internal standard (HMPA) procedure. Hexamethylphosophoroamide was also used as the NMR reference standard. Linear correlations between phosphates and polyphosphate concentrations and ³¹P NMR signal areas were found in the range 81-5236 mg P/dm³, presenting 95-99% recovery and variation coefficient (CV) ≤ 5%. Studied HMPA procedure revealed shorter analysis time and the same recovery (>95%) and precision (CV = 1.3-2.7%) in comparison to MDPA method. Results of phosphate determination by both ³¹P NMR methods were tested against the molybdenumvanadate yellow spectrophotometric method (standard PN-ISO 13730, 1999) using standard reference material (certified phosphate solution).     

Soybean biodiesel methyl esters,free glycerin and acid number quantification by 1H nuclear magnetic resonance spectroscopy

Production of alternative fuels, such as biodiesel, from transesterification of vegetable oil driven by heterogeneous catalysts is a promising alternative to fossil diesel. However, achieving a successful substitution for a new renewable fuel depends on several quality parameters. ¹H NMR spectroscopy was used to determine the amount of methyl esters, free glycerin and acid number in the transesterification of soybean oil with methanol in the presence of hydrotalcite‐type catalyst to produce biodiesel. Reaction parameters, such as temperature and time, were used to evaluate soybean oil methyl esters rate conversion. Temperatures of 100 to 180 °C and times of 20 to 240 min were tested on a 1 : 12 molar ratio soybean oil/methanol reaction. At 180 °C/240 min conditions, a rate of 94.5 wt% of methyl esters was obtained, where free glycerin and free fatty acids were not detected. Copyright © 2012 John Wiley & Sons, Ltd.     

A comparison of quantitative nuclear magnetic resonance methods: internal,external, and electronic referencing

The performance of three quantitative NMR methods was compared in terms of short‐term and long‐term precision and accuracy, robustness, linear range, and general applicability. The Internal Reference method employs a reference material co‐dissolved with sample; the External Reference method employs a reference material contained in a separate solution; and the third method, known as Electronic REference To access In vivo Concentrations (ERETIC), employs an externally calibrated digital reference peak. The Internal Reference method results were the most precise and remained stable within 0.1% for at least 4 weeks. The results from the External Reference and ERETIC methods were practically equivalent to each other during this time. These methods exhibited small differences relative to the standard set by the Internal Reference method and slightly lower precision, establishing them as practical alternatives to the Internal Reference method. In contrast to the Internal Reference method, the External Reference and ERETIC methods possess several advantages that address peak overlap, flexibility of calibration, and duration of applicability. The study was designed such that each spectrum contained the information needed to compare the three methods while all other variables were kept constant. Applicability of pulse width compensation is addressed. ERETIC software compensation and minor adjustments to 90° pulse width were concluded to be unnecessary for this system. Although each of the methods was applied here to specifically calculate and compare chemical purity values, this evaluation applies generally to absolute quantitation by NMR. Copyright © 2013 John Wiley & Sons, Ltd.     

Structure determination of a sildenafil analogue contained in commercial herb drinks

It was found from HPLC analysis that a commercial herb drink contains a putative sildenafil analog. In order to identify the sildenafil-like compound, separation and purification were carried out. The structure determination was performed based on routine 1D and 2D NMR experiments. Unlike sildenafil, the compound contains an ethylpiperazine instead of a methylpiperazine group. In addition, the piperazine and phenyl groups are connected through an acetyl group instead of the sulfonyl group of sildenafil.     

Structural determination of quercusides A and B,new flavonoid glucosides from Quercus incana,by 1D and 2D NMR spectroscopy

Quercusides A and B, new flavonoid glucosides have been isolated from the chloroform soluble fractions of Quercus incana. Their structures were assigned from ¹H and ¹³C nuclear magnetic resonance spectra, distortionless enhancement by polarization transfer (DEPT) and by correlation spectroscopy, heteronuclear multiple quantum correlation (HMQC) and heteronuclear multiple bond correlation (HMBC) experiments. Lupeol, β‐sitosterol and ursolic acid have also been reported from this species. Copyright © 2009 John Wiley & Sons, Ltd.     

5-苄氧亚氨基-5-脱氧阿维菌素B1a的合成

以阿维菌素B1a为原料经过选择性氧化合成5-氧代-5-脱氧阿维菌素B1a, 再与O-取代羟胺盐酸盐反应合成了5个5-苄氧亚氨基-5-脱氧阿维菌素B1a (4a～4e), 通过¹H NMR, ¹³C NMR, MS, IR确证了它们的结构. 生测结果表明它们对于棉铃虫和甜菜夜蛾有一定的杀虫活性.