Quantification of Health-Promoting Compounds by Quantitative

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Chapter 15

Quantification of Health-Promoting Compounds by Quantitative 1HNMR Spectroscopy G. K. Jayaprakasha* and Bhimanagouda S. Patil Vegetable and Fruit Improvement Center, Department of Horticultural Sciences, Texas A&M University, 1500 Research Parkway, A120, College Station, Texas 77845-2119 *E-mail: [email protected].

Nuclear magnetic resonance (NMR) spectroscopy is a wellknown analytical technique for simultaneous quantitation and structural elucidation of small molecules and macromolecules. In natural products research, quantitative NMR (qNMR) gives valuable information on metabolites. qNMR is robust with high precision, good reproducibility and is non-destructive, compared to traditional analytical methods. It has proven to be rapid, highly reliable for the determination of purity, examination of impurities, metabolic profiling, and for the quantitation of single entities in complex mixtures without fractionation or isolation. Therefore, qNMR can be used to simultaneously identify and quantify multiple metabolites. This chapter discusses current challenges in quantitation, metabolomics, and sample preparation, as well as the selection of references for quantitation of health-promoting compounds.

Introduction Nuclear magnetic resonance (NMR) spectroscopy is a well-known analytical technique for elucidating the structure of small molecules and macromolecules (1). NMR spectroscopy uses the application of strong magnetic fields and radio frequency pulses to the nuclei of atoms. For atoms with odd atomic number (1H) or odd mass number (13C), the magnetic field will cause the nucleus to possess spin, which is known as nuclear spin. Absorption of radio frequency energy then © 2014 American Chemical Society In Instrumental Methods for the Analysis and Identification of Bioactive Molecules; Jayprakasha, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


allows the nuclei to be promoted from low-energy to high-energy spin states, and the emission of radiation during the subsequent relaxation process is detected. 1HNMR spectroscopy has been used for quantitative analysis during 1963 for determining the intra-molecular proton ratios (2). In the 1H NMR spectrum, the chemical shift and coupling constants give valuable information about the quantitative relationship between intramolecular and inter-molecular resonances. Also, as long as the analytes contain protons, NMR can analyze any class of compound. Figure 1 depicts the 1HNMR spectrum of L-citrulline in D2O recorded at 400 MHz JEOL ECS spectrometer. The chemical shifts of each signal have been assigned to the respective protons in the L-citrulline molecule. The integral values in each signal denote the number of protons present in molecule.

Figure 1. Structure of L-citrulline and its proton NMR spectrum shows the relative integrals and chemical shifts of intra –molecular resonances. The integral values depends on the number of nuclei per resonance.

In natural products research, quantitative proton nuclear magnetic resonance (qHNMR) has emerged as one of the most reliable, and suitable techniques for comprehensive qualitative and quantitative analysis. The main advantage of qNMR compared to other analytical methods is the primary ratio measurement, since the peak area in qNMR is proportional to the number of nuclei (CH, CH2 and CH3) giving rise to the signal. With qNMR, the quantitation of the compounds present in a complex sample can be performed in a single, rapid, non-destructive measurement. Sample preparation for qNMR is simple and non-tedious and the uncertainty in quantification is minimal (3). NMR spectroscopy has additional advantages, such as the ability to determine molecular structures, the lack of a requirement for individual experimental setup for authentication, validation, and calibration, rapid, and non-destructive measurements. It is also possible to quantitatively analyze multiple metabolites simultaneously from a mixture. 290 In Instrumental Methods for the Analysis and Identification of Bioactive Molecules; Jayprakasha, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


The applicability and ease of use of qNMR has increased due to availability of high-sensitivity detection methods, sample changers with good homogeneity, and modern software packages that allow accurate and precise data processing for large numbers of samples. In the last decade, researchers have reported intensive studies using qNMR for the analysis of individual components in complex mixtures without prior LC separation (4, 5). qNMR does not require a particular reference standard, but quantification can also be performed using an internal standard. Various internal standards have been used in qNMR (Table 1), usually co-dissolved with the sample or introduced in a separate coaxial insert tube.

qNMR and Metabolomics Metabolomics involves the study of small molecules from cells, plants, foods, tissues, organisms or other biological tissues. These small molecules include primary and intermediary metabolites, as well as exogenous compounds, such as secondary metabolites, drugs, and other compounds. Metabolic fingerprinting compares patterns, signatures or fingerprints of metabolites that change in response to external stimuli either in different plant varieties or species, or in response to adulteration, toxins, drug exposure, environmental or genetic alterations (6, 7). Metabolomics fingerprinting is a promising tool in clinical diagnosis, drug screening and toxicology studies (8–12). One of the main problems in metabolomics is the lack of standardized methods, especially for global metabolomics analysis. In analytical chemistry, applications of qNMR include the identification and quantification of targeted and non-targeted metabolites. More recently, qNMR has been used to provide an unbiased view of sample composition, and, simultaneously, to quantify multiple compounds. qNMR has become the method of choice for metabolome studies and quality control of complex natural samples such as foods, plants, herbal remedies, and biofluids (10). Metabolomics is often deemed more discriminating than transcriptomics and proteomics, possibly because there are fewer chemical metabolites than genes and proteins. The main advantages for metabolic profiling using qNMR is faster than proteome and transcriptome analyses, as well as less expensive. Thus, qNMR-based metabolomics presents an ideal choice for systems biology studies on interactions at different molecular levels (13).

Challenges in Metabolomics Analysis The main challenge in metabolomics involves the extraction of metabolites (targeted and non-targeted), detection, identification, and quantification of huge numbers of compounds present in a wide range of concentrations. To address this complex challenge, integration of various analytical platforms including gas chromatography-mass spectrometry (GC-MS), ultra-high performance liquid chromatography combined with MS (UHPLC-MS) and nuclear magnetic resonance (NMR) have been explored to improve metabolite coverage and expand the categories of metabolite identification (14). Moreover, improved 291 In Instrumental Methods for the Analysis and Identification of Bioactive Molecules; Jayprakasha, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


detection techniques (NMR and LC-MS) have been discussed recently for the identification of various metabolites (1, 15). Evident demand exists for the development of robust strategies for preparation of biological samples (16). Due to the heterogeneity of target samples in metabolomics experiments, selection of solvents for the extraction of targeted metabolites and preparation of samples are key steps (17, 18). For example, quantitation of non-polar or mid-polar metabolites requires extraction with deuterated solvents such as CDCl3, CD2Cl2 whereas polar metabolites can be extracted with various solvents such as deuterated water (D2O), CD3OD, CD3CN and DMSO-d6. The liquid samples can be analyzed by lyophilization, followed by extraction with appropriate NMR grade solvents.

Selection of Samples, Storage, and Processing Sampling and sample preparation are critical and valuable steps in metabolite analysis. The sampling time, method of sampling, diurnal, and dietary influences can greatly affect the reproducibility (19, 20) and have major effects on the composition of the metabolome (21, 22). The storage of biological samples is important, as freeze/thaw cycles may have detrimental effects on the stability and composition of the sample (23–25). All these factors will influence the precision, accuracy and reproducibility of results. It seems that the biological variability is always greater than analytical variability, even in studies that use controlled sampling and sample preparation (26). Extra-cellular metabolite samples such as urine, depict a period of metabolic activity and are easy to acquire. Intra-cellular metabolite samples provide a snapshot of the metabolome, but can be time-consuming and difficult to acquire, depending on the accessibility of the tissue. Metabolic processes occur rapidly in biological systems, so both types of samples require quick inhibition of enzymatic processes, generally by freeze-clamping or freezing in liquid nitrogen after harvesting, and subsequent storage at -80°C. However, freezing of biological samples has been reported to cause loss of certain intracellular metabolites (27). To prevent changes in the composition of the samples during storage, the number of cycles of freezing and thawing should be minimized. Acidic treatments (perchloric or nitric acid) may cause severe reduction or degradation of certain metabolites. Physical and chemical disruption of the cells generally involves extraction with polar or non-polar solvents, and distribution of metabolites in polar (methanol/water) and non-polar (chloroform) solvents is also commonly used. Figure 2 shows the extraction efficiency of curcuminoids extracted using CDCl3, acetone-d6 and DMSO-d6. Since curcumin is lipophilic in nature, thus CDCl3 showed selective extraction of curcumin as compared to acetone-d6 and DMSO-d6. The NMR spectra of polar solvents showed other polar compounds signals along with curcumin. All signals were assigned, confirmed and quantitatively determined as curcumin. In case of Figure 2B and 2C, many minor signals denotes the presence of more compounds extracted in these solvnets. Thus selection of solvent is more critical to isolate certain targeted metabolites. 292 In Instrumental Methods for the Analysis and Identification of Bioactive Molecules; Jayprakasha, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


Figure 2. Extraction efficiency of curcuminoids in various solvents such as (A). CDCl3 (B). Acetone – d6 and (C). DMSO-d6 . Sample (525μL) was transferred to an NMR tube and coaxial glass tube containing 60 μL 0.012% 3-(trimethylsilyl) propionic-(2,2,3,3-d4) acid sodium salt in D2O and was inserted into the 5 mm NMR tube. TSP-d4 in reusable external tube served as a quantitative reference. The major compound curcumin signals were assigned.

In some cases, the loss of metabolites occurs due to non-specific binding or adsorption to the container surface; this can be prevented by post-addition of reagents such as bovine serum albumin or Tween-80 (28). Sample collection should be non-invasive and randomized. The choice of sample type and method of sample preparation are critical aspects in metabolomics studies. These aspects directly affect the data quality, accuracy and interpretation of the results. 293 In Instrumental Methods for the Analysis and Identification of Bioactive Molecules; Jayprakasha, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


Sample Preparation

Optimization of metabolites isolation without degradation is a critical step in analytical chemistry. To get satisfactory results, sample preparation should be simple and rapid to prevent metabolite loss, and it should be high-throughput to enable processing of large numbers of samples in short time span. Furthermore, the sample preparation method should be reproducible and include a metabolism-quenching step to represent the true metabolome composition at the time of sampling. One more challenge is that the extraction of metabolites from various matrices, including plants, food, tissues, and organisms, will vary because of the differences in each matrix; thus, extraction procedures need to be optimized. The first step in preparation of biological samples is to freeze at -80°C or in liquid nitrogen, because the pattern of the metabolites may change prior to analysis. Methanol cooling of samples has often been used as a simple and rapid method to terminate metabolism and rupture cells, promoting the release of intracellular metabolites. As the polarity of the solvent increases, the range of metabolites extraction is also increases. Moreover, extractions should be performed at suitable pH, which leads to a maximum recovery of metabolites with minimal extraction interfering materials. The extraction methods must be highly efficient, nonselective, and reproducible, and should not cause degradation of the metabolites. However, the analysis of targeted metabolites or the construction of metabolic profiles requires a selective method of sample preparation to decrease other compounds that may interfere with the analysis.

Reference and Calibration Standards.

As similar to any analytical method, qNMR requires calibration. Most quantitative NMR experiments require referencing of chemical shifts (δ ppm) and quantitation of NMR signals by calibrating sample and standard signals. Table 1 shows the most commonly used standards in qNMR, while TMS and DSS are the IUPAC-approved NMR standards. The referencing and calibration of chemical shifts are often done externally, that is with a separate sample rather than with an internal standard. Chemical shifts can also be calibrated using the residual solvent signal. qNMR requires a well-defined standard material, which is often termed the reference standard. The primary standard should be highly pure, have good solubility, be stable and not be volatile (e.g. TMS). Moreover, it should be a well-characterized material and does not chemically interact with the analyte. In the case of biological samples (e.g., serum, plasma, and other biological fluids), which have abundant proteins, lipoproteins and fatty acids, the selection of internal standard is critical.

294 In Instrumental Methods for the Analysis and Identification of Bioactive Molecules; Jayprakasha, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


Table 1. Structures of Compounds Used for Determination of Concentrations by qNMR and Their Molecular Weights and Chemical Shifts

Quantitation of Health-Promoting Compounds in Food qHNMR has a growing application in analysis of food, because it has numerous advantages over currently used routine analytical methods (29). Current chromatographic methods require method development, optimization of sample preparation, relatively longer analysis times (15-60 min), frequent 295 In Instrumental Methods for the Analysis and Identification of Bioactive Molecules; Jayprakasha, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.

calibrations using identical reference materials. The simplicity and swiftness of qNMR-based methods have been demonstrated to advance food science and technology in the study of metabolic and fermentation processes, composition of foods, or controlling manufacturing stages (29–33). The following two examples were demonstrated for the quantitation of certain health beneficial compounds using qNMR.


Quantitation of Curcumin in Turmeric Samples. Recently, optimized a quantitative proton NMR for the determination of purity of curcuminoids is reported (29). Curcuminoids are yellow pigments with many pharmacological properties including antimicrobial, antiviral, antifungal, anticancer, and anti-inflammatory activities (34). A variety of methods have been reported for the quantification of the curcuminoids, and most of these are spectrophotometric methods, which measure the total color content of the sample (35). Commercial turmeric products contain mixtures of curcumin, demethoxycurcumin, and bisdemethoxycurcumin. However, spectrophotometric methods cannot quantify individual curcuminoids. While HPLC method for the determination of curcuminoids in turmeric was reported (36–38). This HPLC method requires 20 min run time and uses authenticated standards for calibration and quantification. While recently developed qNMR method needed less than 5 min, which can be used for quantitation of large numbers of samples. As an example of the utility of this method, a turmeric powder (50 mg) was extracted in 1 mL of DMSO-d6 for 30 min by sonication at 40 ºC. The sample was filtered though 0.45 micron filter and analyzed by NMR at a frequency of 400 MHz using 5 mm multinuclear inverse probe. A 525 µL DMSO-d6 extracted sample was transferred to a NMR tube. An external coaxial glass tube (Wildmad-LabGlass, Vineland, NJ) containing 60 µL 0.012% 3-(trimethylsilyl) propionic-(2,2,3,3-d4) acid sodium salt (TSP) solution in D2O was inserted into the NMR sample tube as a quantitative reference. The TSP concentration in the tube was pre-calibrated using a separate standard solution. A sufficiently long (16 s) relaxation delay was used to ensure full recovery of magnetization from both sample and internal reference (TSP) for accurate quantization. 1H NMR spectra obtained from the single pulse sequence were used to determine the curcumin content in turmeric sample. The purity of the curcumin in turmeric sample was determined by comparing the peak integrals of the compounds and the reference, taking into account the volume of the sample, the number of protons that contribute to the peak area and the molecular weights of curcumin and the reference standard. Figure 3A shows the 1HNMR spectra of the turmeric sample along with TSP. The TSP signal displayed at δ-0.55 ppm in the sample due to the presence of two solvents in one NMR tube (525 µL of turmeric sample in DMSO-d6 and 60 µL of TSP in D2O). The arrow indicates the signals for curcumin and the rest of the signal for other, minor curcuminoids. Further, the selectivity of each signal was confirmed by spiking a known amount of pure curcumin into the turmeric sample and recording the spectra as described above (Figure 3B). This spectrum clearly shows that the assigned 296 In Instrumental Methods for the Analysis and Identification of Bioactive Molecules; Jayprakasha, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


proton signal intensity is enhanced after spiking with the standard curcumin, which confirms the selectivity of the signal. Using these signal integral values, the purity of compounds was determined using the following formula (39).

Figure 3. 1H NMR signal enhancement by spiking with a known standard and recorded at 400 MHz. (A). Turmeric sample (50 mg) was extracted with 1mL of DMSO-d6 and 600 μL was used, (B). Turmeric sample (400 μL) was spiked with 10mM curcumin (200 μL). The arrows indicates the enhanced curcumin signals.

IA and IRef are the signal integral values of analyte and reference (e.g. TSP), respectively. HA and HRef are the number of protons in analyte and reference, respectively MA and MRef are the molecular weights of analyte and reference, respectively CA is the concentration of analyte or weight of sample and CRef is the concentration of the reference used for the assay. The purity of curcumin (3.8%) was found to be comparable to HPLC analysis with

Quantification of Health-Promoting Compounds by Quantitative

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