Evaluation of the Structural Properties and Isotopic Abundance Ratios of Consciousness Energy Treated Pyridoxine Using LC-MS, GCMS, and NMR Spectroscopy

Pyridoxine (pyridoxol/vitamin B6) is a water-soluble vitamin widely distributed in food and dietary supplements [1]. It occurs naturally in foods such as chickpeas, fish, meat, poultry, nuts, whole grains, bananas, spinach, tofu, avocados, etc. [2] It usually acts like a cofactor or prosthetic group for more than 100 enzymatic reactions. Pyridoxal 5’ phosphate (PLP) and pyridoxamine 5’ phosphate (PMP) are involved in the metabolism of amino acids, carbohydrates, and lipids. Pyridoxine also plays important role in biosynthesis of neurotransmitters and maintaining normal levels of homocysteine, amino acid in the blood, gluconeogenesism, glycogenolysis, immune function (lymphocyte and interleukin-2 production), and hemoglobin formation [3-5]. Pyridoxine hydrochloride is the commonly used salt form of vitamin B6 [6]. Vitamin B6 is generally used as vitamin supplement and also as a component of multivitamin preparations for the prevention and treatment of vitamin B6 deficiency, sideroblastic anaemia, metabolic disorder, cardiovascular disease, Alzheimer’s disease, pyridoxine-dependent epilepsy, pulmonary tuberculosis, hyperhomocysteinaemia, cancer, anxiety, asthma, depression, attention deficit hyperactivity disorder (ADHD), dysmenorrhoea, diabetes, post-partum lactation suppression, McArdle’s disease, osteoporosis, problems from isoniazid, mushroom poisoning, etc. [1,3-8]. Vitamin B6 seldom shows side-effects like headache, sleepiness, numbness, sensory neuropathy (ataxia), etc. It can interact with many medications, i.e., cycloserine, antiepileptic drugs (valproic acid, carbamazepine, phenytoin, etc.), theophylline, etc. might adversely affect vitamin B6 levels [3,5].

There is a quantum energy matrix that surrounds the human body resulting from the continuous movement of the electrically charged particles (ions, cells, etc.) inside the body is called the Biofield Energy [9,10]. Biofield Energy Practitioners have the ability to harness the inherently intelligent energy from the “universal energy field” and can transmit into any living or nonliving object(s) anywhere on the planet. Biofield based Energy Therapies are used against various human disease conditions and accepted in many countries [11,12]. Biofield Energy Healing therapy has been recognized as a Complementary and Alternative Medicine (CAM) health care approach by National Center of Complementary and Integrative Health (NCCIH) with other therapies, medicines and practices such as yoga, Ayurvedic medicine, traditional Chinese herbs and medicines, aromatherapy, Reiki, cranial sacral therapy, Qi Gong, Tai Chi, chiropractic/osteopathic manipulation, meditation, homeopathy, acupressure, acupuncture, healing touch, hypnotherapy, movement therapy, naturopathy, etc. [13,14]. The Trivedi Effect^® is natural and only scientifically proven phenomenon [15]. The Trivedi Effect^®-Biofield Energy Treatment is significantly drawing attention in several fields include materials science [16,17], nutraceuticals [18,19], pharmaceuticals [20,21], organic compounds [22,23], microbiology [24,25], agricultural [26,27], biotechnology [28], medical [29] due to its astounding ability for modification of the characteristic properties. Some of the literature has reported significant problems about the bioavailability of vitamin B6 due to the very limited extent intestinal absorption of B6 vitamers, large rate of elimination due to the non-binding properties of pyridoxine to the proteins of blood plasma, instability of pyridoxine due to the complex formation between various food, drug, and food stored at elevated temperatures [4,6,30,31]. The recent study of the Trivedi Effect^®-Consciousness Energy Treatment on the pharmaceutical/nutraceutical compounds, i.e., resveratrol, berberine, and 25-hydroxyvitamin D3 reported significant improvement of the bioavailability in Male Sprague-Dawley rats [32-34]. These effects of the Energy of Consciousness Treatment (the Trivedi Effect^®) are proposed to act through the possible mediation of neutrinos [15,32-34].

The analysis of stable isotope ratios (e.g., C,H,O,N,S,Fe,Ca,Cu,S, etc.) can provide powerful tracers and chronometers for anthropology, geosciences, archaeology, environmental studies, and health research [35-39]. Highly sophisticated analytical instruments like Gas chromatography – mass spectrometry (GC-MS) and liquid chromatography - mass spectrometry (LC-MS) techniques are widely used for the study of isotope ratio analysis with sufficient precision [38]. So many studies have been performed to evaluate the impact of the Trivedi Effect^® on the isotopic abundance ratios of organic compounds, and the significant alteration of the isotopic abundance ratio of the organic compounds were reported due to the Trivedi Effect^® [22,23]. Thus, the current study has been designed to evaluate influence of the Trivedi Effect^®-Consciousness Energy Treatment on the isotopic abundance ratios P_M+1/P_M (²H/¹H or ¹³C/¹²C or ¹⁵N/¹⁴N or ¹⁷O/¹⁶O) and P_M+2/P_M (¹⁸O/¹⁶O) and structural properties of pyridoxine HCl using LC-MS, GC-MS, and NMR (Nuclear Magnetic Resonance) analytical techniques.

Chemicals and reagents

Pyridoxine hydrochloride (C₈H₁₂ClNO₃; 100%) was purchased from TCI, Japan. All other chemicals used during the experiments were of analytical grade available in India.

Consciousness energy healing treatment strategies

The pyridoxine hydrochloride sample was divided into two parts. One part of the pyridoxine was considered as control/untreated, which was not treated with the Biofield Energy Treatment. The second part of the pyridoxine sample was treated with the Trivedi Effect^®-Consciousness Energy Treatment remotely under standard laboratory conditions for ~3 minutes by Mr. Mahendra Kumar Trivedi and termed as the Biofield Energy Treated/the Trivedi Effect^® Treated pyridoxine. Trivedi was located in the USA, while the test samples were located in the research laboratory in India. This Biofield Energy Treatment was provided through the Mahendra Trivedi’s unique energy transmission process to the test item. Further, the control sample was treated by a “sham” healer for better comparison. The sham healer did not have any knowledge about the Biofield Energy Treatment. The Biofield Energy Treated and untreated samples of pyridoxine were kept in sealed conditions and characterized using LC-MS, GC-MS, and NMR techniques.

Characterization

Liquid chromatography-mass spectrometry (LC-MS) analysis and Calculation of Isotopic Abundance Ratio: The LC-MS analysis of the control and Biofield Energy Treated pyridoxine was performed using LC-Dionex Ultimate 3000, MS-TSQ Endura, (USA) equipped with a photo-diode array (PDA) detector connected with a triple-stage quadrupole mass spectrometer (Thermo Scientific TSQ Endura, USA) with a Thermo Scientific Ion Max NG source and atmospheric pressure chemical ionization (APCI). The analysis was performed on a reversed phase Zorbax Eclipse-C18 100 × 4.6mm, 3.5µm in isocratic mode (A: B-75:25v/v) in the liquid chromatograph. The mobile phase was 0.1% formic acid in water (mobile phase A), and acetonitrile (mobile phase B) at a constant flow rate of 0.5mL/min. The column temperature was kept constant at 35 °C. The injection volume was 5µL and the total run time was 15 minutes. Peaks were monitored using the PDA (295nm) detector. Mass spectrometric analysis was performed under +ve ESI mode. The total ion chromatogram, peak area% and mass spectrum of the individual peak which appeared in LC along with the full scan were recorded. The mass peak intensities of the mass spectrum of the individual peak were recorded.

The natural abundance of C, O, N, and H isotope can be predicted from the comparison of the relative abundance of the isotope peak with respect to the base peak. The values of the natural isotopic abundance of the common elements are obtained from the literature [39-42]. The isotopic abundance ratios (P_M+1/P_M and P_M+2/P_M) for the control and Biofield Energy Treated pyridoxine were calculated.

Percentage (%) change in isotopic abundance ratio of pyridoxine = [(IAR_Treated – IAR_Control)/ IAR_Control) x 100]

Where, IAR_Treated: isotopic abundance ratio in the treated pyridoxine and IAR_Control: isotopic abundance ratio in the control pyridoxine.

Gas chromatography-mass spectrometry (GC-MS) analysis: The GC-MS analysis of the control and the Biofield Energy Treated pyridoxine was performed using Agilent 7890B Gas chromatograph equipped with a silica capillary column HP-5 MS (30m x 0.25mm x 0.25µm) and coupled to a quadrupole detector with pre-filter (5977B, USA) was operated with electron impact (EI) ionization in positive ion mode at 70eV. Oven temperature was programmed from 80 °C (1 min hold) to 200 °C@ 10 °C/min (5min hold) to 300 °C (2min hold) @20 °C/min. Temperatures of the injector, detector (FID), auxiliary, ion source, and quadrupole detector were 250, 260, 280, 230, and 150 °C. Pyridoxine HCl was dissolved in methanol, and 1.0µL was splitlessly injected with helium as a carrier gas with a flow rate of 2.0mL/min. Mass spectra were scanned from m/z 40 to 1050 at a stability of ±1m/z mass accuracy over 48 hours and mass peak intensities of the mass spectrum of the individual peak were recorded.

Percent change in peak intensity (I) was calculated using following equations:

Where, I_Control and I_Treated are the peak intensity of the control and the Biofield Energy Treated samples, respectively.

Nuclear magnetic resonance (NMR) analysis: 1H NMR spectra of pyridoxine HCl were recorded at 400MHz on Agilent-MRDD2 FT-NMR. Approximately 3mg of the sample was dissolved in DMSO-d6. Chemical shifts (d) were in parts per million (ppm) relative to the solvent’s residual proton chemical shift {(CD₃)₂SO, δ = 2.5}. ¹H NMR multiplicities were designated as singlet (s), doublet (d), doublet of doublet (dd), triplet (t), quartet (q), multiplet (m), broad (br), apparent (app). Similarly, ¹³C NMR spectra of pyridoxine HCl were measured at 100MHz on Agilent-MRDD2 FT-NMR spectrometer at room temperature. Approximately 25mg of the sample was dissolved in DMSO-d6. Chemical shifts (d) were in parts per million (ppm) relative to the solvent’s residual carbon chemical shift {(CD₃)₂SO, δ = 39.52}.

Liquid chromatography-mass spectrometry (LC-MS) analysis

The control and the Biofield Energy Treated pyridoxine HCl showed a clear and sharp peak at retention times (Rt) 2.4 minutes in both the chromatograms (Figure 1). The peak area% of the control and the Biofield Energy Treated pyridoxine at Rt 2.4 minutes was 99.99 in case of both control and the Biofield Energy Treated pyridoxine. The peak area% of the Biofield Energy Treated sample was similar to the control sample, which indicated that the polarity of both the samples was similar.

Figure 1:Total ion chromatograms (TIC) of the control and Biofield Energy Treated pyridoxine HCl.

The ESI-MS spectra of the control pyridoxine exhibited the protonated molecular ion mass peak (Figure 2) at m/z 170.02 (calcd for C₈H₁₂NO₃⁺, 170.08). Similarly, the mass spectra of the Biofield Energy Treated pyridoxine exhibited the protonated molecular ion mass peak (Figure 2) at m/z 170.05 (calcd for C₈H₁₂NO₃⁺, 170.08). The parent mass peak at m/z 170 was the base peak which exhibited 100% relative abundance in both the spectra (Figure 2). Although the fragmentation patterns of the control and the Biofield Energy Treated pyridoxine were similar, the relative peak intensities of the Biofield Energy Treated pyridoxine were significantly improved compared to the control sample.

Isotopic abundance ratio analysis

The control and the Biofield Energy Treated samples of pyridoxine showed the protonated molecular ion mass peak at m/z 170.02 (calcd for C₈H₁₂NO₃⁺, 170.08) with 100% relative abundance in the mass spectra. The theoretical calculation of isotopic peak P_M+1 for the protonated pyridoxine presented below:

P (¹³C) = [(8 x 1.1%) x 100% (the actual size of the M⁺ peak)] / 100% = 8.8%

P (²H) = [(12 x 0.015%) x 100%] / 100% = 0.18%

P (¹⁵N) = [(1 x 0.40%) x 100%] / 100% = 0.4%

P (¹⁷O) = [(3 x 0.04%) x 100%] / 100% = 0.12%

P_M+1 i. e.¹³C, ²H, ¹⁵N, and ¹⁷O contributions from C₈H₁₂NO₃+ to m/z 171 = 9.5%

Similarly, the theoretical calculation of isotopic peak P_M+2 for the protonated pyridoxine presented below:

P (¹⁸O) = [(3 x 0.20%) x 100%] / 100% = 0.6%

P_M+2 of 18O contribution from C₈H₁₂NO₃+ to m/z 172 = 0.6%

The calculated isotopic abundance of P_M+1 and P_M+2 values were near to the observed values (Table 1). The probability of A + 1 and A + 2 elements having an isotope with one and two mass unit heavier, respectively than the most abundant isotope (i.e., ¹³C, ¹⁵N, ¹⁷O, and ¹⁸O) contributions to the mass of the isotopic molecular ion [M+1]⁺ and [M+2]⁺. ²H did not contribute much any isotopic m/z ratios because of its less natural abundance compared to the abundances of C, N, and O isotopes [40-43]. But, the contributions of ¹³C, ¹⁵N, ¹⁷O, and ¹⁸O was major from pyridoxine to the isotopic mass peak at m/z 171 and 172 confirmed from the calculations. Therefore, P_M, P_M+1, and P_M+2 of the pyridoxine at m/z 170, 171, and 172 of the control and Biofield Energy Treated samples were obtained from the experimental relative abundance of M⁺, (M+1)⁺, and (M+2)⁺ peaks, respectively in the mass spectra (Figure 2 and Table 1).

Figure 2:The ESI-MS spectra of the control and the Biofield Energy Treated pyridoxine at Rt 2.4 minutes in the chromatograms.

Table 1:LC-ESI-MS isotopic abundance ratio analysis of control and Biofield Energy Treated pyridoxine.

P_M = the relative peak intensity of the parent molecular ion M⁺; P_M+1 = the relative peak intensity of the isotopic molecular ion [M+1]⁺, P_M+2 = the relative peak intensity of the isotopic molecular ion [M+2]⁺, and M = mass of the parent pyridoxine molecule.

Table 1 summarized the percentage change in the isotopic abundance ratios (P_M+1/P_M and P_M+2/P_M) in the Biofield Energy Treated pyridoxine compared to the control sample. The isotopic abundance ratio of P_M+1/P_M (²H/¹H or ¹³C/¹²C or ¹⁵N/¹⁴N or ¹⁷O/¹⁶O) in the Biofield Energy Treated pyridoxine was increased by 11.92% compared to the control sample (Table 1). This indicated that the ²H, ¹³C, ²H, ¹⁵N, and ¹⁷O contributions from C₈H₁₂NO₃⁺ to the isotopic m/z 171 in the Biofield Energy Treated pyridoxine sample was significantly increased compared to the control sample. Correspondingly, the isotopic abundance ratio of P_M+2/P_M (¹⁸O/¹⁶O) in the Biofield Energy Treated pyridoxine also significantly increased by 153.85% compared to the control sample (Table 1). Therefore, the ¹⁸O contribution from C₈H₁₂NO₃⁺ to the isotopic m/z 172 in the Biofield Energy Treated pyridoxine was significantly increased compared to the control sample.

The significant improvement of the isotopic abundance ratios (PM+1/PM and PM+2/PM) might be the impact of the Trivedi Effect^®-Consciousness Energy Treatment which purports to provide the necessary energy for the neutrino oscillations which leads to the alteration of the fundamental properties of any subject(s) [15,22,23]. The neutrino oscillations seem to give credence to the postulates of researchers on The Trivedi Effect^® [15]. A neutrino is an electrically neutral elementary particle with negligible unit mass, interacts only via the weak subatomic force and gravity [44,45]. Alteration in atomic/molecular level postulated to the changes in atomic mass and charge through the possible mediation of neutrinos [15,46-48]. The altered isotopic abundance ratio leads to the alteration of the kinetic isotope effects of the atoms/molecules. This is very useful to study the reaction mechanism, understand the enzymatic transition state, and enzyme mechanism, which is supportive to design effective and specific inhibitors, etc. [39]. Therefore, the Biofield Energy Treated pyridoxine with altered isotopic abundance ratios was assumed to be more advantageous for the designing of better nutraceutical/ pharmaceutical formulations.

Gas chromatography-mass spectrometry (GC-MS) analysis

The GC-MS chromatograms of the control and the Biofield Energy Treated samples of pyridoxine are shown in Figure 3. Both the chromatograms showed a similar type of chromatographic peaks with similar Rt. The retention times of pyridoxine was found at 13.8 minutes in both the chromatograms of control and the Biofield Energy Treated samples. This indicated that the polarity of the control and the Biofield Energy Treated pyridoxine is same. But, the GC chromatographic peak area% of the Biofield Energy Treated pyridoxine (70.21%) was significantly increased by 15.88% compared to the control sample (60.59%) (Table 2). This indicated that the solubility of the Biofield Energy Treated pyridoxine was increased compared to the control sample.

Figure 3:GC chromatograms of the control and Biofield Energy Treated pyridoxine HCl.

Table 2:GC-MS chromatographic and mass spectra analysis at Rt 13.8 minutes of the control and the Biofield Energy Treated pyridoxine.

The GC-MS spectra of the control and Biofield Energy Treated pyridoxine at Rt of 13.8 minutes exhibited the presence of the molecular ion (Figure 4) at m/z 169.1 (calcd for C₈H₁₁NO₃⁺, 169.07). The other mass fragmentation peak at lower m/z 151.1, 136.0, 122.1, 106.1, and 94.1 corresponded to the molecular formula C₈H₉NO₂^•+, C₈H₁₀NO⁺, C₇H₈NO⁺, C₇H₈N⁺, and C₆H₈N⁺, respectively were observed in both control and the Biofield Energy Treated pyridoxine (Figures 4 & 5). The mass fragmentation patterns of the Biofield Energy Treated pyridoxine were similar compared to the control sample. But the mass peak intensities of the Biofield Energy Treated pyridoxine were significantly altered compared to the control pyridoxine. The mass peak intensity of the control and the Biofield Energy Treated pyridoxine were 62965.86 and 83278.98, respectively at Rt 13.8 minutes. The mass peak intensity of the Biofield Energy Treated pyridoxine was significantly increased by 32.26% compared to the control pyridoxine (Table 2). The improved mass peak intensities were assumed to be the influence of the Trivedi Effect^®-Consciousness Energy Treatment [15,22,23].

Figure 4:GC-MS spectra of the control and Biofield Energy Treated pyridoxine at Rt 13.8 minutes.

Figure 5:Proposed fragmentation pathway of pyridoxine.

Nuclear magnetic resonance (NMR) spectroscopy analysis

¹H spectra of the control and the Biofield Energy Treated pyridoxine are shown in Figure 6. The signals for the protons coupling of CH₃, CH₂, CH, and OH protons in both the ¹H NMR spectra of pyridoxine were in the range of d 2.59 to 16.03ppm (Table 3). The proton signals of the Biofield Energy Treated pyridoxine were close to the control sample. The 13C NMR spectra of the control and the Biofield Energy Treated pyridoxine are shown in Figure 7. The carbon signals for CH₃, CH₂, CH, =C=, and C-OH groups in both the control and the Biofield Energy Treated ¹³C NMR spectra were in the range of 14.7-151.89 (Table 3). The experimental data closely matched to the published literature [49,50]. The ¹H and ¹³C NMR results indicated that there was no structural modification of the Biofield Energy Treated pyridoxine compared to the control sample.

Figure 6:The ¹H NMR spectra of the control and Biofield Energy Treated pyridoxine.

Figure 7:The ^{13C NMR spectra of the control and Biofield Energy Treated pyridoxine.}

Table 3:¹H and ^{13C NMR spectroscopic data of both the control and Biofield Energy Treated pyridoxine.}

s: singlet; b: broad.

The Trivedi Effect^®-Consciousness Energy Treatment on pyridoxine by Mr. Mahendra Kumar Trivedi showed an astounding significant impact on the relative peak intensities, peak area%, and isotopic abundance ratios. The LC-ESI-MS analysis of both the control and the Biofield Energy Treated pyridoxine samples showed the protonated parent ion mass at m/z 170 (calcd for C₈H₁₂NO₃⁺, 170.08) at retention times (Rt) 2.41 minutes. But, the relative peak intensities of the Biofield Energy Treated pyridoxine were significantly improved compared to the control sample. The isotopic abundance ratios of P_M+1/P_M (²H/¹H or ¹³C/¹²C or ¹⁵N/¹⁴N or ¹⁷O/¹⁶O) and P_M+2/P_M (¹⁸O/¹⁶O) were significantly increased by 11.92% and 153.85%, respectively in the Biofield Energy Treated pyridoxine compared to the control sample. Thus, ²H, ¹³C, ²H, ¹⁵N, ¹⁷O contributions from C₈H₁₂NO₃⁺ to the isotopic m/z 171 and ¹⁸O contribution to the isotopic m/z 172 significantly increased in the Biofield Energy Treated sample compared with the control sample. The GC chromatographic peak area% and GC-MS mass peak intensity of the Biofield Energy Treated sample significantly increased by 15.88% and 32.26%, respectively compared to the control sample. The proton and carbon signals for CH₃, CH₂, NH₂, CH, =C=, and C-OH groups in the ¹H and ¹³C NMR spectra of the control and the Biofield Energy Treated samples were similar. The increased mass peak intensities, peak area%, and isotopic abundance ratios in the Biofield Energy Treated pyridoxine might be the impact of the Trivedi Effect^®-Consciousness Energy Treatment via the possible mediation of neutrinos, which further lead to the change in the kinetic isotope effects of the Biofield Energy Treated pyridoxine. Thus, the Trivedi Effect^® Treated pyridoxine could be advantageous for designing better nutraceutical, dietary supplements and/or pharmaceutical formulations which might provide better therapeutic response against vitamin B6 deficiency, hereditary sideroblastic anemia, premenstrual syndrome, cardiovascular disease, pyridoxine-dependency seizures, febrile seizures, pulmonary tuberculosis, metabolic disorders, Alzheimer’s disease, cancer, hyperhomocysteinemia, anxiety, asthma, depression, attention deficit hyperactivity disorder (ADHD), dysmenorrhea, akathisia, angioplasty, birth outcomes, cognitive function, hyperkinetic cerebral dysfunction syndrome, carpal tunnel syndrome, hypertension, immune system function, McArdle’s disease, osteoporosis, lactation suppression, pregnancy-induced nausea and vomiting, breast pain, Tardive dyskinesia, autism, stroke recurrence, problems from isoniazid, mushroom poisoning, etc.

The authors are grateful to GVK Biosciences Pvt. Ltd., Trivedi Science, Trivedi Global, Inc., and Trivedi Master Wellness for their assistance and support during this work.

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