Pulse Length Based Concentration Determination Research Articles

Quantification of sesquiterpene pyridine alkaloids from genus Tripterygium by band-selective HSQC NMR

Sesquiterpene pyridine alkaloids (SPAs) are bioactive analogues derived from the genus Tripterygium and have anti-inflammatory and anti-rheumatic properties. Attributed to the similar sesquiterpene structures, the total SPAs showed severe peak overlap in 1D NMR and HPLC, leading to difficulties in identification and quantification. Interestingly, the application of band-selective HSQC NMR that specifically excited the region corresponding to the H-3 of SPAs prompted a signal separation of the total SPAs. Based on the high resolution, 23 SPAs were identified from the band-selective HSQC spectrum. The coupling constants (JCH, JHH) and relaxation times (T1, T2) of SPAs were measured, and it was found that they caused less than 1% attenuation of the HSQC signals, so the HSQC signals of SPAs had almost uniform responses. The concentrations of 23 SPAs were determined by standard curve method, using wilforgine as the calibration. In addition, we extended the pulse length-based concentration determination (PULCON) as a more efficient external standard method to the band-selective HSQC spectrum, and the results showed that the concentrations of alkaloids determined by PULCON were consistent with those measured by standard curve method. The developed quantification approach was validated according to the <761> of United States Pharmacopoeia (USP), demonstrating that the established band-selective HSQC approach is reliable for the rapid quantification of analogues in botanical extracts.

Two Ways to Achieve the Same Goal-Two Validated Quantitative NMR Strategies for a Low-Abundance Natural Product in Standardized Extracts: The Case of Hepatodamianol in Turnera diffusa.

The quantification of low-abundance secondary metabolites in plant extracts is an analytical problem that can be addressed by different analytical platforms, the most common being those based on chromatographic methods coupled to a high-sensitivity detection system. However, in recent years nuclear magnetic resonance (NMR) has become an analytical tool of primary choice for this type of problem because of its reliability, inherent simplicity in sample preparation, reduced analysis time, and low solvent consumption. The versatility of strategies based on quantitative NMR (qNMR), such as internal and external standards and electronic references, among others, and the need to develop validated analytical methods make it essential to compare procedures that must rigorously satisfy the analytical well-established acceptance criteria for method validation. In this work, two qNMR methods were developed for the quantification of hepatodamianol, a bioactive component of T. diffusa. The first method was based on a conventional external standard calibration, and the second one was based on the pulse length-based concentration determination (PULCON) method using the ERETIC2 module as a quantitation tool available in TopSpin software. The results show that both procedures allow the content of the analyte of interest in a complex matrix to be determined in a satisfactory way, under strict analytical criteria. In addition, ERETIC2 offers additional advantages such as a reduction in experimental time, reagent consumption, and waste generated.

A Simple and Feasible Quantification of Metabolites in the Human Follicular Fluid Using 1H HR-MAS NMR Spectroscopy

This study presents a reliable method for the quantification of metabolite concentrations in follicular fluid with the high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy and the ERETIC2 (Electronic REference To access In vivo Concentrations) based on PULCON (pulse length based concentration determination) principle. The positive effect of the HR-MAS probe technology on spectral quality and its ability to perform analyses with very low sample amounts were the most important factors of proposing this method. In evaluating the performance of the proposed method, standard creatine solutions in different concentrations containing DSS (2,2-dimethyl-2-silapentane-5-sulfonate sodium salt) as an internal reference standard were analyzed using different pulse programs (cpmgpr1d and zg30). The results obtained with the ERETIC2 were compared with the classical internal standard NMR quantification method (DSS method). The relative standard deviation (RSD) values for ERETIC2 were in the range of 0.3% - 5.7% and recovery values were calculated as minimum 90.3%, while RSD values for DSS method were in the range of 0.1% - 3.1% and recovery values were minimum 97.0%. Besides, it was observed that the metabolite concentration values calculated using the ERETIC2 procedure of follicular fluid samples obtained from the women with endometriosis and healthy controls were compatible with the values those obtained using different methodologies. The obtained results showed that the proposed quantification method based on the HR-MAS spectroscopy can easily be used in biological fluids and therefore it can be utilized as a good alternative to the internal standard method considering its accuracy and precision.

A Quantitative 1H NMR Method for Screening Cannabinoids in CBD Oils.

Toxicologically relevant levels of the psychoactive ∆9-tetrahydocannabinol (∆9-THC) as well as high levels of non-psychoactive cannabinoids potentially occur in CBD (cannabidiol) oils. For consumer protection in the fast-growing CBD oil market, facile and rapid quantitative methods to determine the cannabinoid content are crucial. However, the current standard method, i.e., liquid chromatography combined with tandem mass spectrometry (HPLC-MS/MS), requires a time-consuming multistep sample preparation. In this study, a quantitative nuclear magnetic resonance spectroscopy (qNMR) method for screening cannabinoids in CBD oils was developed. Contrary to the HPLC-MS/MS method, this qNMR features a simple sample preparation, i.e., only diluting the CBD oil in deuterochloroform. Pulse length-based concentration determination (PULCON) enables a direct quantification using an external standard. The signal intensities of the cannabinoids were enhanced during the NMR spectra acquisition by means of multiple suppression of the triglycerides which are a major component of the CBD oil matrix. The validation confirmed linearity for CBD, cannabinol (CBN), ∆9-THC and ∆8-THC in hemp seed oil with sufficient recoveries and precision for screening. Comparing the qNMR results to HPLC-MS/MS data for 46 commercial CBD oils verified the qNMR accuracy for ∆9-THC and CBD, but with higher limits of detection. The developed qNMR method paves the way for increasing the sample throughput as a complementary screening before HPLC-MS/MS.

Determination of Sample Concentrations by PULCON NMR Spectroscopy.

PULCON (Pulse Length Based Concentration Determination) is a powerful, versatile, non-invasive, and accurate technique for measuring solution concentrations during routine NMR spectroscopy. As solutes are quantified directly by their unique resonances, this technique avoids weight-based errors caused by contaminants (e.g. moisture), allows NMR samples to be directly employed in biological assays, and is particularly useful for quantifying small molecules, peptides, unstable molecules, and other materials that are difficult to weigh or handle. This article provides an introductory guide for biological and medicinal chemists, and highlights the diversity of applications.

Application of 1H Nuclear Magnetic Resonance Spectroscopy as Spirit Drinks Screener for Quality and Authenticity Control.

Due to legal regulations, the rise of globalised (online) commerce and the need for public health protection, the analysis of spirit drinks (alcoholic beverages >15% vol) is a task with growing importance for governmental and commercial laboratories. In this article a newly developed method using nuclear magnetic resonance (NMR) spectroscopy for the simultaneous determination of 15 substances relevant to assessing the quality and authenticity of spirit drinks is described. The new method starts with a simple and rapid sample preparation and does not need an internal standard. For each sample, a group of 1H-NMR spectra is recorded, among them a two-dimensional spectrum for analyte identification and one-dimensional spectra with suppression of solvent signals for quantification. Using the Pulse Length Based Concentration Determination (PULCON) method, concentrations are calculated from curve fits of the characteristic signals for each analyte. The optimisation of the spectra, their evaluation and the transfer of the results are done fully automatically. Glucose, fructose, sucrose, acetic acid, citric acid, formic acid, ethyl acetate, ethyl lactate, acetaldehyde, methanol, n-propanol, isobutanol, isopentanol, 2-phenylethanol and 5-(hydroxymethyl)furfural (HMF) can be quantified with an overall accuracy better than 8%. This new NMR-based targeted quantification method enables the simultaneous and efficient quantification of relevant spirit drinks ingredients in their typical concentration ranges in one process with good accuracy. It has proven to be a reliable method for all kinds of spirit drinks in routine food control.

Evaluating the effect of roasting on coffee lipids using a hybrid targeted-untargeted NMR approach in combination with MRI

An integrated targeted-untargeted 1H and 13C Nuclear Magnetic Resonance (NMR) analysis was applied to determine the impact of roasting on coffee lipids. For targeted analysis, both an internal standard (IS) method, as well as the ERETIC2 tool based on PULCON (Pulse Length-based Concentration determination), were used for quantitation. PULCON allows for quantitative analysis without sample contamination with an IS and was found to be in very good agreement with the traditional IS approach as indicated by a systematic Bland-Altman comparison study. For the untargeted analysis, NMR was coupled with multivariate statistical analysis (MVSA), namely Principal Component Analysis (PCA), Hierarchical Cluster Analysis (HCA), and Orthogonal Projection to Latent Structures Discriminant Analysis (OPLS-DA). 13C NMR spectra were acquired using a z-stored spin-echo sequence to achieve higher spectral quality, which is important for both targeted and untargeted analysis. Results showed that roasting has a clear effect on coffee lipids, with diterpenes, oxidation/hydrolysis products and unsaturated fatty acid chains being the most significant markers. In addition, the application of MRI indicated important morphological alterations in bean structure and lipid migration from the endosperm to the surface of the coffee bean.

Utilization of 31P PULCON for Quantitative Hydroxyl Group Determination in Lignin by NMR Spectroscopy

The amount of hydroxyl groups is a crucial parameter when characterizing lignin as a raw material and when developing lignin-based applications. Currently, the most common method used for quantitative hydroxyl group determination is 31P nuclear magnetic resonance (NMR) spectroscopy after phosphitylation of the hydroxyl groups. The method relies on an internal standard for the quantification. The NMR signals of the internal standard have been shown to either overlap with signals from the lignin or to have poor stability due to degradation of the internal standard. To overcome these drawbacks, we have used PULCON (pulse length-based concentration determination) for the quantification of phosphitylated lignin. The method is based on the fact that the product of the intensity of an NMR signal and the length of the 90° pulse is directly proportional to the concentration of the sample. We conclude that the PULCON method could be used for hydroxyl group determination of lignin, and the results obtained are compa...

A validated NMR method for the quantitative determination of rebaudioside A in commercial sweeteners

In recent years, the demand for rebaudioside A (Reb A) in the food industry has increased due to its sweetening power and organoleptic properties. Currently, there is a large variety of food products on the market for human consumption containing Reb A, and among them, sweeteners stand out. In the present work, a strategy for the quantification of Reb A in commercial sweeteners using quantitative 1H nuclear magnetic resonance (qHNMR) with an external standard method applying the pulse length–based concentration determination (ERETIC2) is described. The developed qHNMR method notably satisfies the validation parameters, such as linearity, precision, accuracy, reproducibility, robustness, limit of detection, and limit of quantification (LOQ). The lineal range was established between 0.3–14.8 mM. The LOQ (0.3 mM) was sufficiently low to assess the quantification of Reb A in analysed samples. Intraday and intermediate precision were evaluated in different matrices and the percent relative standard deviation (%RSD) founded were lower than 1.93, and 2.79, respectively. Additionally, this qHNMR procedure has advantages such as a fast analysis time, easy sample preparation and low generation of solvent waste, which represents an environmentally responsible analytical alternative for the determination of Reb A in commercial sweeteners.

Evaluation of mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH) in pure mineral hydrocarbon-based cosmetics and cosmetic raw materials using 1H NMR spectroscopy.

Mineral hydrocarbons consist of two fractions, mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH). MOAH is a potential public health hazard because it may include carcinogenic polycyclic compounds. In the present study, 400 MHz nuclear magnetic resonance (NMR) spectroscopy was introduced, in the context of official controls, to measure MOSH and MOAH in raw materials or pure mineral hydrocarbon final products (cosmetics and medicinal products). Quantitative determination (qNMR) has been established using the ERETIC methodology (electronic reference to access in vivo concentrations) based on the PULCON principle (pulse length based concentration determination). Various mineral hydrocarbons (e.g., white oils, paraffins or petroleum jelly) were dissolved in deuterated chloroform. The ERETIC factor was established using a quantification reference sample containing ethylbenzene and tetrachloronitrobenzene. The following spectral regions were integrated: MOSH δ 3.0 - 0.2 ppm and MOAH δ 9.2 - 6.5, excluding solvent signals. Validation showed a sufficient precision of the method with a coefficient of variation <6% and a limit of detection <0.1 g/100 g. The applicability of the method was proven by analysing 27 authentic samples with MOSH and MOAH contents in the range of 90-109 g/100 g and 0.02-1.10 g/100g, respectively. It is important to distinguish this new NMR-approach from the hyphenated liquid chromatography-gas chromatography methodology previously used to characterize MOSH/MOAH amounts in cosmetic products. For mineral hydrocarbon raw materials or pure mineral hydrocarbon-based cosmetic products, NMR delivers higher specificity without any sample preparation besides dilution. Our sample survey shows that previous methods may have overestimated the MOAH amount in mineral oil products and opens new paths to characterize this fraction. Therefore, the developed method can be applied for routine monitoring of consumer products aiming to minimize public health risks.

Evaluation of mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH) in pure mineral hydrocarbon-based cosmetics and cosmetic raw materials using 1H NMR spectroscopy.

Mineral hydrocarbons consist of two fractions, mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH). MOAH is a potential public health hazard because it may include carcinogenic polycyclic compounds. In the present study, 400 MHz nuclear magnetic resonance (NMR) spectroscopy was introduced, in the context of official controls, to measure MOSH and MOAH in raw materials or pure mineral hydrocarbon final products (cosmetics and medicinal products). Quantitative determination (qNMR) has been established using the ERETIC methodology (electronic reference to access in vivo concentrations) based on the PULCON principle (pulse length based concentration determination). Various mineral hydrocarbons (e.g., white oils, paraffins or petroleum jelly) were dissolved in deuterated chloroform. The ERETIC factor was established using a quantification reference sample containing ethylbenzene and tetrachloronitrobenzene. The following spectral regions were integrated: MOSH δ 3.0 - 0.2 ppm and MOAH δ 9.2 - 6.5, excluding solvent signals. Validation showed a sufficient precision of the method with a coefficient of variation <6% and a limit of detection <0.1 g/100 g. The applicability of the method was proven by analysing 27 authentic samples with MOSH and MOAH contents in the range of 90-109 g/100 g and 0.02-1.10 g/100g, respectively. It is important to distinguish this new NMR-approach from the hyphenated liquid chromatography-gas chromatography methodology previously used to characterize MOSH/MOAH amounts in cosmetic products. For mineral hydrocarbon raw materials or pure mineral hydrocarbon-based cosmetic products, NMR delivers higher specificity without any sample preparation besides dilution. Our sample survey shows that previous methods may have overestimated the MOAH amount in mineral oil products and opens new paths to characterize this fraction. Therefore, the developed method can be applied for routine monitoring of consumer products aiming to minimize public health risks.

Quantitative Nuclear Magnetic Resonance Spectroscopy Based on PULCON Methodology: Application to Quantification of Invaluable Marine Toxin, Okadaic Acid.

ERETIC2 (Electronic Reference To access In vivo Concentrations 2) based on PULCON (Pulse Length–based Concentration determination) methodology is a quantitative NMR (qNMR) using an external standard. The performance of the PULCON method was assessed using maleic acid (MA). Quantification of the diarrhetic shellfish toxin and okadaic acid by PULCON was successfully consistent with that obtained by a conventional internal standard method, demonstrating that the PULCON method is useful for the quantification of invaluable marine toxins without any contaminations by an internal standard.

Automated Multicomponent Analysis of Soft Drinks Using 1D 1H and 2D 1H-1H J-resolved NMR Spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy is evaluated as an efficient analytical technique for simultaneous identification and quantitation of multiple compounds in alcohol-free beverages. The method was validated for three different types of soft drinks, including a beverage with high sugar content, a diet product and an energy drink. The sample preparation required only 600 μL of degassed soft drink, 70 μL of 0.1 % TSP-d4 in D2O (for referencing and locking) and 100 μL of phosphate buffer (pH = 2.89). The simultaneous quantitative analysis of eleven compounds, such as sugars, flavourings, sweeteners, organic acids, alcohol (ethanol), vitamins and amino acids, was achieved using an automated procedure based on the PULCON principle (pulse length based concentration determination). Recovery rates were between 80 % (HMF) and 106 % (ethanol). The limits of quantitation (LOQ) varied between 52 mg/L for saccharin and 12 g/L for sucrose due to different matrix contents. Sample analysis time is about 25 min including sample preparation and automated data processing. A comparison with conventional methods showed the suitability of NMR for routine analysis of soft drinks.

A simple and accurate protocol for absolute polar metabolite quantification in cell cultures using quantitative nuclear magnetic resonance

Absolute analyte quantification by nuclear magnetic resonance (NMR) spectroscopy is rarely pursued in metabolomics, even though this would allow researchers to compare results obtained using different techniques. Here we report on a new protocol that permits, after pH-controlled serum protein removal, the sensitive quantification (limit of detection [LOD] = 5−25 μM) of hydrophilic nutrients and metabolites in the extracellular medium of cells in cultures. The method does not require the use of databases and uses PULCON (pulse length-based concentration determination) quantitative NMR to obtain results that are significantly more accurate and reproducible than those obtained by CPMG (Carr–Purcell–Meiboom–Gill) sequence or post-processing filtering approaches. Three practical applications of the method highlight its flexibility under different cell culture conditions. We identified and quantified (i) metabolic differences between genetically engineered human cell lines, (ii) alterations in cellular metabolism induced by differentiation of mouse myoblasts into myotubes, and (iii) metabolic changes caused by activation of neurotransmitter receptors in mouse myoblasts. Thus, the new protocol offers an easily implementable, efficient, and versatile tool for the investigation of cellular metabolism and signal transduction.

Standardless multicomponent qNMR analysis of compounds with overlapped resonances based on the combination of ICA and PULCON.

A fast and reliable nuclear magnetic resonance (NMR) method for quantitative analysis of targeted compounds with overlapped signals in complex mixtures has been established. The method is based on the combination of chemometric treatment for spectra deconvolution and the PULCON principle (pulse length based concentration determination) for quantification. Independent component analysis (ICA) (mutual information least dependent component analysis (MILCA) algorithm) was applied for spectra deconvolution in up to six component mixtures with known composition. The resolved matrices (independent components, ICs and ICA scores) were used for identification of analytes, calculating their relative concentrations and absolute integral intensity of selected resonances. The absolute analyte concentrations in multicomponent mixtures and authentic samples were then calculated using the PULCON principle. Instead of conventional application of absolute integral intensity in case of undisturbed signals, the multiplication of resolved IC absolute integral and its relative concentration in the mixture for each component was used. Correction factors that are required for quantification and are unique for each analyte were also estimated. The proposed method was applied for analysis of up to five components in lemon and orange juice samples with recoveries between 90% and 111%. The total duration of analysis is approximately 45 min including measurements, spectra decomposition and quantification. The results demonstrated that the proposed method is a promising tool for rapid simultaneous quantification of up to six components in case of spectral overlap and the absence of reference materials.

Determination of the purity of pharmaceutical reference materials by 1H NMR using the standardless PULCON methodology

A fast and reliable nuclear magnetic resonance spectroscopic method for quantitative determination (qNMR) of targeted molecules in reference materials has been established using the ERETIC2 methodology (electronic reference to access in vivo concentrations) based on the PULCON principle (pulse length based concentration determination).The developed approach was validated for the analysis of pharmaceutical samples in the context of official medicines control, including ibandronic acid, amantadine, ambroxol and lercanidipine. The PULCON recoveries were above 94.3% and coefficients of variation (CVs) obtained by quantification of different targeted resonances ranged between 0.7% and 2.8%, demonstrating that the qNMR method is a precise tool for rapid quantification (approximately 15min) of reference materials and medicinal products. Generally, the values were within specification (certified values) provided by the manufactures. The results were in agreement with NMR quantification using an internal standard and validated reference HPLC analysis.The PULCON method was found to be a practical alternative with competitive precision and accuracy to the classical internal reference method and it proved to be applicable to different solvent conditions. The method can be recommended for routine use in medicines control laboratories, especially when the availability and costs of reference compounds are problematic.

Accurate Determination of Reference Materials and Natural Isolates by Means of Quantitative 1H NMR Spectroscopy

A fast and precise proton nuclear magnetic resonance (qHNMR) method for the quantitative determination of low molecular weight target molecules in reference materials and natural isolates has been validated using ERETIC 2 (Electronic REference To access In vivo Concentrations) based on the PULCON (PULse length based CONcentration determination) methodology and compared to the gravimetric results. Using an Avance III NMR spectrometer (400 MHz) equipped with a broad band observe (BBO) probe, the qHNMR method was validated by determining its linearity, range, precision, and accuracy as well as robustness and limit of quantitation. The linearity of the method was assessed by measuring samples of l-tyrosine, caffeine, or benzoic acid in a concentration range between 0.3 and 16.5 mmol/L (r(2) ≥ 0.99), whereas the interday and intraday precisions were found to be ≤2%. The recovery of a range of reference compounds was ≥98.5%, thus demonstrating the qHNMR method as a precise tool for the rapid quantitation (~15 min) of food-related target compounds in reference materials and natural isolates such as nucleotides, polyphenols, or cyclic peptides.

Concentration measurements by PULCON using X-filtered or 2D NMR spectra

Sample concentrations can be measured by nuclear magnetic resonance (NMR) spectroscopy without an internal reference compound using pulse length based concentration determination (PULCON) with 1D NMR spectra. PULCON delivers most accurate results if the spectrum of the sample of interest contains a resolved resonance; but can also be applied to spectral regions with overlapping resonances. If the exact number of lines contributing to the overlapping spectral region is not known, a corresponding error makes the result less precise. The uncertainty about the number of contributing resonances can be reduced with experiments that discriminate different classes of resonances by filtering techniques or by extending PULCON to two-dimensional NMR spectra. We demonstrate the application of PULCON with a 1D 15N-filtered experiment where aromatic resonances of a protein can be observed without interference from 15N-bound protons. Further, we extend PULCON to 2D NMR spectra that permits to determine the exact number of resonances. This extension can readily be applied with sample that contain different solutes.