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

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Advances
in
Bioscience
and
Bioengineering
2017; 5(1): 1-11
http://www.sciencepublishinggroup.com/j/abb
doi: 10.11648/j.abb.20170501.11
ISSN: 2330-4154 (Print); ISSN: 2330-4162 (Online)
Evaluation of Isotopic Abundance Ratio and Structural
Properties of Magnesium Gluconate After Treatment with
the Energy of Consciousness Using LC-MS and NMR
Spectroscopy
Mahendra Kumar Trivedi
1
, Alice Branton
1
, Dahryn Trivedi
1
, Gopal Nayak
1
,
Cathryn Dawn Nykvist
1
, Celine Lavelle
1
, Daniel Paul Przybylski
1
, Dianne Heather Vincent
1
,
Dorothy Felger
1
, Douglas Jay Konersman
1
, Elizabeth Ann Feeney
1
, Jay Anthony Prague
1
,
Joanne Lydia Starodub
1
, Karan Rasdan
1
, Karen Mie Strassman
1
, Leonid Soboleff
1
,
Maire Anne Mayne
1
, Mary M. Keesee
1
, Padmanabha Narayana Pillai
1
, Pamela Clarkson Ansley
1
,
Ronald David Schmitz
1
, Sharyn Marie Sodomora
1
, Kalyan Kumar Sethi
2
, Parthasarathi Panda
2
,
Snehasis Jana
2, *
1
Trivedi Global, Inc., Henderson, Nevada, USA
2
Trivedi Science Research Laboratory Pvt. Ltd., Bhopal, Madhya Pradesh, India
Email address:
publication@trivedieffect.com (S. Jana)
*
Corresponding author
To cite this article:
Mahendra Kumar Trivedi, Alice Branton, Dahryn Trivedi, Gopal Nayak, Cathryn Dawn Nykvist, Celine Lavelle, Daniel Paul Przybylski,
Dianne Heather Vincent, Dorothy Felger, Douglas Jay Konersman, Elizabeth Ann Feeney, Jay Anthony Prague, Joanne Lydia Starodub,
Karan Rasdan, Karen Mie Strassman, Leonid Soboleff, Maire Anne Mayne, Mary M. Keesee, Padmanabha Narayana Pillai, Pamela Clarkson
Ansley, Ronald David Schmitz, Sharyn Marie Sodomora, Kalyan Kumar Sethi, Parthasarathi Panda, Snehasis Jana. Evaluation of Isotopic
Abundance Ratio and Structural Properties of Magnesium Gluconate After Treatment with the Energy of Consciousness Using LC-MS and
NMR Spectroscopy. Advances in Bioscience and Bioengineering. Vol. 5, No. 1, 2017, pp. 1-11. doi: 10.11648/j.abb.20170501.11
Received: January 31, 2017; Accepted: February 13, 2017; Published: February 25, 2017
Abstract:
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
(P
M+1
/P
M
) 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 R
t
of 1.81 and 2.06 min, whereas the treated sample
displayed peaks at R
t
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, CH
2
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 P
M+1
/P
M
(
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 P
M+1
/P
M
(
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.
Keywords:
Biofield Energy Healing Treatment, Energy of Consciousness Healing Treatment, Biofield Energy Healers,
The Trivedi Effect
®
, Magnesium Gluconate, LC-MS, NMR, Isotopic Abundance Ratio, Isotope Effects
1. 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,
Alzheimers disease, asthma, pre-eclampsia and eclampsia,
cancer, etc. [4-8]. 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-
20], biotechnology [21-23], materials science [24-28],
pharmaceutical sciences [29-31], medical science [32, 33],
and microbiology [34-36]. 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
C/
12
C or
17
O/
16
O or
25
Mg/
24
Mg) in both of the
Biofield Energy Treated and untreated samples was
targeted to evaluate the influence of Biofield Energy
Healing Treatment on the isotopic abundance ratio in
magnesium gluconate.
Advances in Bioscience and Bioengineering 2017; 5(1): 1-11 3
2. Materials and Methods
2.1. 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.
2.2. 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 Healers 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.
2.3. Characterization
2.3.1. 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.
2.3.2. 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 [43-45] are presented in
Table 1.
Table 1. The isotopic composition (the natural isotopic abundance) of the
elements.
Element (A)
Symbol
Mass
% Natural
Abundance
A + 1 Factor
A + 2 Factor
Hydrogen
1
H 1 99.9885
2
H
2 0.0115 0.015n
H
Carbon
12
C 12 98.892
13
C
13 1.108 1.1n
C
Oxygen
16
O 16 99.762
17
O 17 0.038 0.04n
O
18
O 18 0.200 0.20n
O
Magnesium
24
Mg 24 78.99
25
Mg 25 10.00 12.66 n
Mg
26
Mg 26 11.01 13.94 n
Mg
A: Element; n: no of H, C, O, Mg, etc.
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
C,
1
H,
16
O,
24
Mg, etc.) contributions to the mass of the parent
4 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
molecular ion [M
+
].
P
M+1
represents the relative peak intensity of the isotopic
molecular ion [(M+1)
+
] expressed in percentage
= (no. of
13
C x 1.1%) + (no. of
2
H x 0.015%) + (no. of
17
O
x 0.04%) + (no. of
25
Mg x 12.66%)
i.e. the probability to have A + 1 element (for e.g.
13
C,
2
H,
25
Mg, etc.) contributions to the mass of the isotopic
molecular ion [(M+1)
+
]
P
M+2
represents the relative peak intensity of the isotopic
molecular ion [(M+2)
+
] expressed in the percentage
= (no. of
18
O x 0.20%) + (no. of
26
Mg x 13.94%)
i.e. the probability to have A + 2 elements (for e.g.
18
O,
26
Mg, etc.) contributions to the mass of isotopic molecular
ion [(M+2)
+
]
Isotopic abundance ratio (IAR) for A + 1 elements = P
M +
1
/P
M
Similarly, isotopic abundance ratio of A + 2 elements =
P
M+2
/P
M
Percentage (%) change in isotopic abundance ratio =
[(IAR
Treated
- IAR
Control
)/ IAR
Control
) x 100] (1)
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.
2.3.3. 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].
3. Results and Discussion
3.1. 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.
Figure 1. Total ion chromatograms (TIC) of the control and Biofield Energy Treated magnesium gluconate using The Waters
®
ACQUITY UPLC.
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) indicated the presence of the mass for
protonated magnesium gluconate ion at m/z 415 [M + H]
+
(calculated for C
12
H
23
MgO
14
+
, 415). Adduct of the
magnesium gluconate with methanol (pseudo molecular ion)
was found at m/z 447 [M + CH
3
OH + H]
+
(calculated for
C
13
H
27
MgO
15
+
, 447). Adducts formation, either with solvents
(i.e. methanol), alkali or metal ions or other contaminating
components are frequently observed in the ESI analysis [47,
48].
Advances in Bioscience and Bioengineering 2017; 5(1): 1-11 5
Figure 2. The ESI-MS (+ve ion mode) spectra of the control and Biofield Energy Treated magnesium gluconate.
Figure 3. The ESI-MS (-ve ion mode) spectra of the control and Biofield Energy Treated magnesium gluconate.
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.
The notable fragment ion peaks in the lower region of the
molecular ion at m/z 415 were observed in the ESI-MS
spectrum of the control sample at m/z 380, 279, 272, 255,
206, 190, 161, 153, 147, 135, 121, and 105, due to the
successive removal of water and alkyl groups from [M + H]
+
and consequently, the internal molecular rearrangement,
corresponded to the following proposed molecular formula
C
12
H
20
MgO
12
+
, C
8
H
15
MgO
9
+
, C
9
H
12
MgO
8
+
, C
8
H
7
MgO
8
+
,
C
8
H
6
MgO
5
2+
, C
6
H
6
O
7
+
, C
6
H
9
O
5
+
, C
5
H
13
O
5
+
, C
5
H
7
O
5
+
,
C
5
H
11
O
4
+
, C
4
H
9
O
4
+
, and C
4
H
9
O
3
+
, 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 pseudo-
molecular ion was at m/z 613 (calcd for C
18
H
37
MgO
21
4+
,
613). The second pseudo-molecular ion was due to the
adduct formation with methanol at m/z 643 (calcd for
C
19
H
38
MgO
22
+
, 643). The last pseudo-molecular ion was at
m/z 665 (calcd for C
19
H
37
Mg
2
O
22
+
, 665) due to the adduct
formation of C
18
H
33
MgO
21
with one Mg
++
atom as well as
with methanol. Consequently, the characteristic fragment ion
peaks in the ESI-MS spectrum of Biofield Energy Treated
sample were observed at m/z 387, 357, 284, 272, 219, 197,
179, 147, 133, 115, and 100, which corresponded to the
following proposed molecular formula C
12
H
27
MgO
12
+
,
C
11
H
25
MgO
11
+
, C
8
H
20
MgO
9
+
, C
9
H
12
MgO
8
+
, C
6
H
11
MgO
7
+
,
C
6
H
13
O
7
+
, C
6
H
11
O
6
+
, C
5
H
7
O
5
+
, C
5
H
9
O
4
+
, C
4
H
3
O
4
+
, and
C
4
H
4
O
3
2+
respectively (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
H
35
MgO
21
+
and C
19
H
38
MgO
22
+
that was due to the magnesium gluconate
chelate with one gluconate ion.
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