Evaluation of Isotopic Abundance Ratio and Structural Properties of Magnesium Gluconate After Treatment with the Energy of Consciousness Using LC-MS and NMR Spectroscopy

The current research work was aimed to investigate the impact of The Trivedi Effect Energy of Consciousness Healing Treatment (Biofield Energy Healing) on the structural properties and isotopic abundance ratio (PM+1/PM) of magnesium gluconate using LC-MS and NMR spectroscopy. Magnesium gluconate was divided into two parts. One part was denoted as the control, while the another part was defined as The Trivedi Effect ® Treated sample, which received the Biofield Energy Healing Treatment remotely from eighteen renowned Biofield Energy Healers. The total ion chromatogram of the control sample showed two peaks at Rt of 1.81 and 2.06 min, whereas the treated sample displayed peaks at Rt of 1.79 and 2.04 min. The ESI-MS spectra of the control and the treated samples revealed the presence of the mass for magnesium gluconate ion in two forms at m/z 447 (adduct form with methanol) and 415 (protonated ion) in positive ionization mode. But, it showed the mass for the gluconate ion at m/z 195 in the negative ionization mode. The fragmentation pattern of magnesium gluconate in the treated sample was notably altered compared with the control sample. The proton and carbon signals for CH, CH2 and CO groups in the proton and carbon NMR spectra were found almost similar for the control and treated samples. The LC-MS based isotopic abundance ratio analysis indicated that the PM+1/PM ( 2 H/ 1 H or 13 C/ 12 C or 17 O/ 16 O or 25 Mg/ 24 Mg) in the treated magnesium gluconate ion at m/z 415 was significantly decreased by 79.24% compared with the control sample. Similarly, the isotopic abundance ratio of PM+1/PM ( 2 H/ 1 H or 13 C/ 12 C or 17 O/ 16 O) in the gluconate ion was decreased by 6.62% in the treated sample compared with the control sample. The treated magnesium gluconate might be beneficial in the nutraceutical and/or pharmaceutical industries for designing various forms of formulations, which could be providing better therapeutic response against various diseases such as diabetes mellitus, allergy, aging, inflammatory diseases, immunological disorders, and other chronic infections. Consequently, The Trivedi Effect ® Energy of Consciousness Healing Treated 2 Mahendra Kumar Trivedi et al.: Evaluation of Isotopic Abundance Ratio and Structural Properties of Magnesium Gluconate After Treatment with the Energy of Consciousness Using LC-MS and NMR Spectroscopy magnesium gluconate would be more helpful for understanding the enzymatic reactions as well as assist in the designing of the novel potent enzyme inhibitors by applying its kinetic isotope effects.


Introduction
Magnesium ion (Mg 2+ ) is a major intracellular divalent cation and an essential mineral for several enzymes, DNA and RNA synthesis, reproduction and protein synthesis. It is also a vital coherent controller of glycolysis and the Krebs cycle [1,2]. Magnesium gluconate (C 12 H 22 MgO 14 ) is the organometallic salt of magnesium with gluconic acid produced from glucose catalyzed by glucose oxidase [3]. Magnesium gluconate is found to be a more powerful antioxidant than other magnesium salts and it is useful for the prevention and treatment of many diseases such as cardiovascular diseases, diabetes mellitus, allergy, inflammatory diseases, immunological disorders, Alzheimer's disease, asthma, pre-eclampsia and eclampsia, cancer, etc. [4][5][6][7][8]. It can be used as neuroprotective [9], for the treatment of oxidative stress induced ischemia/reperfusion injury [10] and also labor in women arrested initially with intravenous therapy as an oral tocolytic agent [11]. Magnesium gluconate showed the highest bioavailability and most physiologically acceptable salt among other magnesium salts like chloride, sulfate, carbonate, acetate, citrate, lactate, aspartate, etc. [8,12]. Therefore, magnesium gluconate was considered as one of the components in a novel proprietary herbomineral formulation for the source of magnesium ion. This herbomineral formulation which is designed as nutraceutical supplement can be used for the prevention and treatment of various human diseases.
Since ancient times, many different cultures, religions and systems of belief have recognized a living force that preserves and inhabits every living organism. This force is the source of 'life' and has been called various names, such as prana by the Hindus, qi or chi by the Chinese, and ki by the Japanese. This is believed to co-relate with the soul, spirit and mind. This hypothetical vital force has been scientifically evaluated and is now considered the Bioenergetics Field. The Biofield Energy is a dynamic electromagnetic field surrounding the human body, resulting from the continuous emission of low-level light, heat, and acoustical energy from the body. Biofield Energy is infinite, para dimensional and can freely flow between the human and environment [13,14]. So, a human has the ability to harness energy from the ionosphere of the earth, the "universal energy field", and transmit it to any living organism (s) or nonliving object (s) around the globe. The object or recipient always receives the energy and responds in a useful way. This process is known as The Trivedi Effect ® -Biofield Energy Healing Treatment [15,16]. Biofield (Putative Energy Field) based Energy Therapies are used worldwide to promote health and healing. The National Center of Complementary and Integrative Health (NCCIH) has recognized and accepted Biofield Energy Healing as a Complementary and Alternative Medicine (CAM) health care approach in addition to other therapies, medicines and practices such as natural products, deep breathing, yoga, Tai Chi, Qi Gong, chiropractic/osteopathic manipulation, meditation, massage, special diets, homeopathy, progressive relaxation, guided imagery, acupressure, acupuncture, relaxation techniques, hypnotherapy, healing touch, movement therapy, pilates, rolfing structural integration, mindfulness, Ayurvedic medicine, traditional Chinese herbs and medicines, naturopathy, essential oils, aromatherapy, Reiki, cranial sacral therapy and applied prayer (as is common in all religions, like Christianity, Hinduism, Buddhism and Judaism) [17]. Biofield Energy Treatment (The Trivedi Effect ® ) has been extensively studied with significant outcomes in many scientific fields such as agriculture [18][19][20], biotechnology [21][22][23], materials science [24][25][26][27][28], pharmaceutical sciences [29][30][31], medical science [32,33], and microbiology [34][35][36]. Literature demonstrated that Biofield Energy Healing Treatment (The Trivedi Effect ® ) might be an alternative method for increasing or decreasing the natural isotopic abundance ratio of the substances [37,38]. The stable isotope ratio analysis has the wide applications in several scientific fields for understanding the isotope effects resulting from the variation of the isotopic composition of the molecule [39,40]. Conventional mass spectrometry (MS) techniques such as liquid chromatography -mass spectrometry (LC-MS), gas chromatography -mass spectrometry (GC-MS) are widely used for isotope ratio analysis with sufficient precision [41]. Hence, LC-MS and NMR (Nuclear Magnetic Resonance) were used in this study to characterize the structural properties of the Biofield Energy Treated and untreated magnesium gluconate qualitatively for the purpose of the pharmaceutical and nutraceutical industrial applications. Consequently, LC-MS based isotopic abundance ratio analysis of P M+1 /P M ( 2 H/ 1 H or 13

Chemicals and Reagents
Magnesium gluconate hydrate was procured from Tokyo Chemical Industry Co., Ltd. (TCI), Japan. All other chemicals used in the experiment were of analytical grade available in India.

Energy of Consciousness Treatment Strategies
Magnesium gluconate was one of the components of the new proprietary herbomineral formulation, which was developed by our research team and was used per se as the test compound for the current study. The test compound was divided into two parts. One part of the test compound did not receive any sort of treatment and defined as untreated or control magnesium gluconate sample. Consequently, another part of the test compound was treated with The Trivedi Effect ® -Energy of Consciousness Healing Treatment remotely by eighteen renowned Biofield Energy Healers and defined as Biofield Energy Treated magnesium gluconate. Eleven of the Biofield Energy Healers were located in the U. S. A., four in Canada, one in Ireland, one in the United Kingdom, and one in Russia performed the Biofield Energy Treatment on the test compound that was located in the research laboratory of GVK Biosciences Pvt. Ltd., Hyderabad, India. The Biofield Energy Treatment was provided for 5 minutes through the Healer's Unique Energy Transmission process remotely to the test compound, which was kept under laboratory conditions. None of the Biofield Energy Healers in this study visited the laboratory in person, nor had any contact with the compounds. Similarly, the control compound was subjected to a "sham" healer for 5 minutes, under the same laboratory conditions. The sham healer did not have any knowledge about the Biofield Energy Treatment. After that, the Biofield Energy Treated and untreated samples were kept in similar sealed conditions. The control and Biofield Energy Treated samples were analyzed by LC-MS and NMR spectroscopy.

Liquid Chromatography-Mass Spectrometry (LC-MS) Analysis
Liquid chromatography was accomplished using The Waters ® ACQUITY UPLC, Milford, MA, USA equipped with a binary pump (The Waters ® BSM HPLC pump), autosampler, column heater and a photo-diode array (PDA) detector. The column used for the study was a reversed phase Acquity BEH shield RP C18 (150 X 3.0 mm, 2.5 µm). The column temperature was kept constant at 40°C. The mobile phase was 2mM ammonium acetate in water as mobile phase A and acetonitrile as mobile phase B. Chromatographic separation was achieved with the following gradient program: 0 minute -5%B; 1 minute -5%B; 15 minutes -97%B; 20 minutes -97%B; 21 minutes -5%B; 25 minutes -5%B. The flow rate was at a constant flow rate of 0.4 mL/minutes. The control and Biofield Energy Treated samples were dissolved in a mixture of water and methanol (60:40 v/v) to prepare a 1 mg/mL stock solution. An aliquot of 2 µL of the stock solution was used for analysis by LC-ESI-MS and the total run time was 25 minutes.
Mass spectrometric analysis was accompanied on a Triple Quad (Waters Quattro Premier XE, USA) mass spectrometer equipped with an electrospray ionization (ESI) source with the following parameters: electrospray capillary voltage 3.5 kV; source temperature 100°C; desolvation temperature 350°C; cone voltage 30 V; desolvation gas flow 1000 L/h and cone gas flow 60 L/h. Nitrogen was used in the electrospray ionization source. The multiplier voltage was set at 650 V. LC-MS was taken in positive and negative ionization mode and with the full scan (m/z 50-1400). The total ion chromatogram, peak area% and mass spectrum of the individual peak (appeared in LC) were recorded.

Isotopic Abundance Ratio Analysis
The isotopic abundance ratio of the control and Biofield Energy Treated magnesium gluconate was performed. The natural abundance of each isotope can be predicted from the comparison of the height of the isotope peak with respect to the base peak, i.e. relative abundance in the mass spectra [37,38,42,43]. The natural isotopic abundance of the elements are obtained from several literatures [43][44][45] are presented in Table 1. The following method was used for calculating the isotopic abundance ratio: P M stands for the relative peak intensity of the parent molecular ion [M + ] expressed in percentage. In other way, it indicates the probability to have A element (for e.g. 12 Where, IAR Treated is isotopic abundance ratio in the treated magnesium gluconate sample and IAR Control is isotopic abundance ratio in the control magnesium gluconate sample.

Nuclear Magnetic Resonance (NMR) Analysis
1 H NMR spectra were recorded in a 400 MHZ VARIAN FT-NMR spectrometer at room temperature. Data refer to solutions in D 2 O with the residual solvent protons as internal references. 1 H NMR multiplicities were designated as singlet (s), doublet (d), triplet (t), multiplet (m), and broad (br). 13 C NMR spectra were measured at 100 MHz on a VARIAN FT-NMR spectrometer at room temperature. Chemical shifts (δ) were in parts per million (ppm) relative to the solvent's residual proton chemical shift (D 2 O, δ = 4.65 ppm) and solvent's residual carbon chemical shift (D 2 O, δ = 0 ppm) [46].

Liquid Chromatography-Mass Spectrometry (LC-MS) Analysis
The total ion chromatograms (TIC) of the control and Biofield Energy Treated magnesium gluconate are shown in Figure 1. The TIC of the control magnesium gluconate showed two peaks at retention time (R t ) of 1.81 and 2.06 minutes with the peak area% of 61.22% and 38.78%, respectively in the chromatogram. Similarly, the chromatogram of the Biofield Energy Treated magnesium gluconate exhibited two peaks at R t of 1.79 and 2.04 min with the peak area% of 61.77% and 38.23%, respectively. The results indicated that Biofield Energy did not show any impact in the alteration of the polarity/affinity of the Biofield Energy Treated magnesium gluconate compared with the control sample. The ESI-MS spectra of both the control and Biofield Energy Treated samples are presented in Figure 2 and 3. The ESI-MS (+ve ion mode) spectra of both the control and Biofield Energy Treated magnesium gluconate at R t of 2.1 minutes (Figure 2) [47,48].  The pseudo molecular ion magnesium gluconate at m/z 447 displayed 100% relative intensity in the control sample, while the fragment ion peak at m/z 100 exhibited the 100% relative peak intensity in the Biofield Energy Treated sample. , respectively as shown in Figure 4. The peak at m/z 431, which was due to the magnesium gluconate adduct with the ammonium ion was only found in the control sample. This ammonium adduct formation might be due to the usage of ammonium acetate with water as mobile phase [47,48]. The other major ions in the higher m/z region of the control ESI-MS spectrum ( Figure 2) were found at m/z 613, 643 and 665. These mass indicated for the mass of the magnesium gluconate chelate with one gluconate ion through coordinate covalent bond (C 18 H 33 MgO 21 , 609) as shown in Figure 4. But it existed in three different pseudo-molecular ions. First pseudomolecular ion was at m/z 613 (calcd for C 18 (Figure 4). The prominent ions observed in the higher m/z region of the Biofield Energy Treated ESI-MS spectrum ( Figure 2) were at m/z 611 and 643 corresponding to the molecular formula C 18   -, 391) both in the control and Biofield Energy Treated samples. Overall, from the above data, it can be concluded that the fragmentation pattern of the Biofield Energy Treated magnesium gluconate was significantly altered compared with the control sample. From the analysis, the pseudo molecular ion formation found less in case of the Biofield Energy Treated sample compared with the control sample in solution.

Isotopic Abundance Ratio Analysis
The molecular formula of magnesium gluconate is ) showing 53.31% (control) and 48.73% (Biofield Energy Treated) relative intensity. The relative abundances of an isotopic peak are the contributions of several different isotopes and its composition to the same peak [43]. The theoretical calculation of P M+1 for the protonated magnesium gluconate was presented as below: 27% Thus, the probability to A + 1 elements having an isotope that has one mass unit heavier than the most abundant isotope (i.e. 13 C, 2 H, 17 O, and 25 Mg) contributions to the mass of the isotopic molecular ion [(M+1) + ]. In general, the deuterium did not contribute much to any isotopic mass, because the natural abundance of deuterium is too small relative to the natural abundances of carbon, oxygen and magnesium isotopes [43][44][45]. Hence, 13 C and 25 Mg have the major contributions from magnesium gluconate and magnesium gluconate to the isotopic peaks. The percentage change in isotopic abundance ratio of P M+1 /P M for magnesium gluconate in the Biofield Energy Treated and control samples are presented in Table 2. The isotopic abundance ratio of P M+1 /P M in the Biofield Energy Treated magnesium gluconate was decreased by 79.24% compared with the control sample (Table 2). 13 C and 25 Mg contributions from C 12 H 23 MgO 14 + to m/z 415 in the Biofield Energy Treated sample were reduced compared with the control sample. The calculated abundance of P M+1 (7.05%) in magnesium gluconate closely matched to the experimental value obtained in the control sample ( Table 3). The isotopic abundance ratio of P M+1 /P M in the gluconate ion was significantly decreased by 6.62% in the Biofield Energy Treated sample compared with the control sample. Hence, 13 25 Mg/ 24 Mg) in the Biofield Energy Treated magnesium gluconate was significantly decreased compared with the control sample. The alteration in the isotopic abundance ratio could be due to the Biofield Energy Healing Treatment.
Mass spectroscopic analysis of the several organic compounds revealed that the isotopic abundance of [M+1] + ions were decreased, thereby suggesting the change in number of neutrons in the molecule. It was then postulated to the alterations in atomic mass and atomic charge through the possible mediation of neutrino oscillation [52][53][54]. Thus, it is assumed that The Trivedi Effect ® -Biofield Energy Healing might provide the required energy for the neutrino oscillations. The changes of neutrinos inside the molecule in turn modified the particle size, chemical reactivity, density, thermal behavior, selectivity, binding energy, etc. [44,54].
Kinetic isotope effect that is resultant from the variation in the isotopic abundance ratio of one of the atoms in the reactants in a chemical reaction is very useful to study the reaction mechanism as well as for understanding the enzymatic transition state and all aspects of enzyme mechanisms that is supportive for designing enormously effective and specific inhibitors [41,49,51]. As magnesium is an essential cofactor for various enzymatic reactions, the Biofield Energy Treated magnesium gluconate that had altered isotopic abundance ratio might be useful for the study of enzyme mechanism as well as assist in the designing of novel potent enzyme inhibitors.

Nuclear Magnetic Resonance Spectroscopy ( 1 H and 13 C NMR) Analysis
The 1 H and 13 C NMR spectra of the control and Biofield Energy Treated magnesium gluconate are shown in Figures 5a-d. NMR assignments of the control and Biofield Energy Treated magnesium gluconate are presented in the Table 5. The structure of magnesium gluconate contain a large number of OH groups and the hydroxyl protons have no signal in the 1 H NMR spectra because these protons are replaced by deuterium when deuterated water (D 2 O) was used as solvent for spectra recording The signals for the protons signals for CH 2 and CH groups of the control and Biofield Energy Treated magnesium gluconate were in the range of δ 3.54-4.06 ppm and δ 3.54-4.07 ppm respectively, which complies the reported proton spectrum of sodium gluconate [46]. The signals for the carbon signals for CO, CH 2, and CH groups in the 13 C NMR spectra of the control and Biofield Energy Treated magnesium gluconate were in the range of δ 62.60-178.44 ppm and δ 62.43-178.44 ppm, respectively. Thus, the proton and carbon signals for CH, CH 2 and CO groups in the proton and carbon NMR spectra of the control and the Biofield Energy Treated samples were found almost similar. It was then concluded that the structure of the magnesium gluconate was remained unaltered due to the Biofield Energy Healing Treatment.

Conclusions
LC-ESI-MS analysis indicated that magnesium gluconate in the solution might be existed in situ of three forms, magnesium gluconate, gluconic acid and a chelate form of magnesium gluconate with the gluconic acid by the coordinate covalent bond. The total ion chromatogram (TIC) of the control sample showed two peaks at R t of 1.81 and 2.06 minutes, whereas The Trivedi Effect ® treated sample displayed these peaks at R t of 1.79 and 2.04 minutes. The ESI-MS spectra of the control and the treated samples revealed the presence of the mass for magnesium gluconate ion in two forms at m/z 447 (adduct form with methanol) and 415 (protonated ion) in positive ionization mode. But, it showed the mass for the gluconate ion at m/z 195 in the negative ionization mode. The fragmentation pattern of magnesium gluconate in the treated sample was significantly altered as compared to the control sample. Consequently, the LC-ESI-MS based isotopic abundance ratio analysis indicated that the P M+1 /P M ( 2 H/ 1 H or 13  ) + to m/z 196 was also decreased in the treated sample in comparison with the control sample. The treated sample might exhibit isotope effects such as altered physicochemical and thermal properties, rate of the reaction, selectivity and binding energy due to its changed isotopic abundance ratio of P M+1 /P M compared with the control sample. The treated magnesium gluconate might be helpful to understand the enzymatic reactions as well as design the novel potent enzyme inhibitors by using its kinetic isotope effects. Besides, The Trivedi Effect ® -Energy of Consciousness Healing Treatment, could be a useful approach in the design of better nutraceutical and/or pharmaceutical formulations that can offer significant therapeutic responses against various diseases such as diabetes mellitus, allergies and septic shock, stress-related disorders like sleep disorder, insomnia, anxiety, depression, Attention Deficit Disorder (ADD), Attention Deficit Hyperactive Disorder (ADHD), mental restlessness (mind chattering), brain frog, low libido, impotency, lack of motivation, mood swings, fear of the future, confusion, migraines, headaches, forgetfulness, overwhelm, loneliness, worthlessness, indecisiveness, frustration, irritability, chronic fatigue, obsessive/compulsive behavior and panic attacks, inflammatory diseases and immunological disorders like Lupus, Systemic Lupus Erythematosus, Hashimoto Thyroiditis, Type 1 Diabetes, Asthma, Chronic peptic ulcers, Tuberculosis, Hepatitis, Chronic active hepatitis, Celiac