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Special Issue 5 • 2015
J Chromatograph Separat Techniq
ISSN:2157-7064 JCGST, an open access journal
Research Article Open Access
Trivedi et al., J Chromatograph Separat Techniq 2015, S6
http://dx.doi.org/10.4172/2157-7064.S6-003
Special Issue Open Access
Chromatography
Separation Techniques
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ISSN: 2157-7064
Investigation of Isotopic Abundance Ratio of Biofield Treated Phenol
Derivatives Using Gas Chromatography-Mass Spectrometry
Trivedi MK1, Branton A1, Trivedi D1, Nayak G1, Saikia G2 and Jana S2*
1Trivedi Global Inc., 10624 S Eastern Avenue Suite A-969, Henderson, NV 89052, USA
2Trivedi Science Research Laboratory Pvt. Ltd., Hall-A, Chinar Mega Mall, Chinar Fortune City, Hoshangabad Rd., Bhopal, Madhya Pradesh, India
*Corresponding author: Snehasis Jana, Trivedi Science Research Laboratory
Pvt. Ltd., Hall-A, Chinar Mega Mall, Chinar Fortune City, Hoshangabad Rd.,
Bhopal-462 026, Madhya Pradesh, India, Tel: +91-755-6660006; Fax: +91-755-
6660006; E-mail: publication@trivedisrl.com
Received August 27, 2015; Accepted September 14, 2015; Published September
24, 2015
Citation: Trivedi MK, Branton A, Trivedi D, Nayak G, Saikia G, et al. (2015)
Investigation of Isotopic Abundance Ratio of Bioeld Treated Phenol Derivatives
Using Gas Chromatography-Mass Spectrometry. J Chromatograph Separat
Techniq S6:003. doi:10.4172/2157-7064.S6-003
Copyright: © 2015 Trivedi MK, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Abstract
Butylatedhydroxytoluene (BHT) and 4-methoxyphenol (4-MP) are phenol derivatives that are generally known
for their antioxidant properties and depigmenting activities. The aim of this study was to evaluate the impact of
bioeld energy treatment on the isotopic abundance in BHT and 4-MP using gas chromatography-mass spectrometry
(GC-MS). BHT and 4-MP samples were divided into two parts: control and treated. The control group remained
untreated while the treated group was subjected to Mr. Trivedi’s bioeld treatment. Control and treated samples
were characterized using GC-MS. The GC-MS data revealed that the isotopic abundance ratio of 13C/12C or 2H/1H
(PM+1)/PM and 18O/16O (PM+2)/PM increased signicantly in treated BHT and 4-MP (where PM- primary molecule,
PM+1- isotopic molecule either for 13C or 2H and PM+2 is the isotopic molecule for 18O). The isotopic abundance ratio
of (PM+1)/PM in the treated BHT and 4-MP was increased up to 181.27% and 380.73% respectively as compared
to their respective control. Moreover, the isotopic abundance ratio of (PM+2)/PM in the treated BHT and 4-MP
increased up to 185.99% and 355.33% respectively. GC-MS data suggests that the bioeld treatment signicantly
increased the isotopic abundance of 2H, 13C and 18O in the treated BHT and 4-MP as compared to the control.
Keywords: Bioeld energy treatment; Butylatedhydroxytoluene;
Gas chromatography-mass spectrometry; 4-methoxyphenol; Isotopic
abundance
Abbreviations
GC-MS: Gas Chromatography-Mass spectrometry; PM: Primary
Molecule; PM+1: Isotopic molecule either for 13C/12C or 2H/1H; PM+2:
Isotopic molecule for 18O/16O; BHT: Butylatedhydroxytoluene; 4-MP:
4-methoxyphenol
Introduction
Butylatedhydroxytoluene (BHT) is a crystalline stable solid, but it
is light-sensitive and reactive to acid chlorides, acid anhydrides, and
oxidizing agents. BHT is used as an antioxidant in many products
including food, pharmaceuticals, rubbers, paint, and petroleum
products [1]. Its antioxidant mechanism was well studied and divided
into two steps. In the rst step, it forms a stable phenoxyl radical,
which further forms the parent and a quinone methide (QM) [2].
QM is a reactive electrophilic species that easily forms adducts with
nucleophiles or polymerize in the next step. Adduct formation between
the nucleophilic groups of the active pharmaceutical ingredients with
QM in pharmaceutical formulations is very much possible [3]. In
food preservation, BHT reacts with atmospheric oxygen preferentially
rather than oxidizing food materials, thereby protecting them from
decomposition. It is used to preserve food odor, color, and avor [1].
4-methoxyphenol (4-MP) is a common active pharmaceutical
ingredient in topical drugs used for skin depigmentation.
4-hydroxyanisole and the retinoid tretinoin have been used individually
as depigmenting agents. 4-MP is oen mixed with tretinoin, a topical
retinoid [4]. A common formulation for this drug is an ethanolic
solution of 2% 4-MP and 0.01% tretinoin by mass. BHA is a commercial
mixture of mono- and di-tert-butylated products of 4-MP, which is an
eective anti-oxidant with maximum carry-through protection used in
foodstus [5] and pharmaceuticals [6]. Many polymerization inhibitors
(e.g., phenols) work best in the presence of oxygen because they
intercept peroxyl radicals and decelerate oxygen consumption while
stopping chain propagation. 4-MP is an inhibitor of this type. 4-MP
has tremendous ability to quench peroxyl radicals and alkyl radicals
via hydrogen abstraction mechanism, which leads to the formation of
a phenoxyl radical [7,8]. e phenoxyl radical is less reactive because
it is stabilized by the resonance eect. e antimicrobial activity of
phenolic compounds is highly dependent upon the chemical structure
of the molecules [9]. e bactericidal actions of substituted phenols
and the normal alkyl derivatives of p-chlorophenols were examined
against Gram-negative and Gram-positive bacteria [10,11]. e
antibacterial potential of essential oils of sweet basil Ocimum basilicum
L. (Lamiaceae) containing 1.8% 4-MP and methanol extracts was
evaluated for controlling the growth range of food-borne pathogenic
bacteria [12]. It was reported that BHT at high levels as well as at lower
levels found in foods might have anti-cancer properties, possibly by
damaging free radicals or by stimulating the production of enzymes
that detoxify carcinogens. On the other hand 4-MP is carcinogenic to
the fore-stomach [13,14].
e rate of chemical reaction depends on the mass of the nucleus
with dierent isotopic substitutions, which slightly aect the partitioning
of energy within molecules. ese deviations from perfect chemical
equivalence are termed as isotope eects. e isotopic abundance ratio
is commonly reported in terms of atom percent.
Citation: Trivedi MK, Branton A, Trivedi D, Nayak G, Saikia G, et al. (2015) Investigation of Isotopic Abundance Ratio of Bioeld Treated Phenol
Derivatives Using Gas Chromatography-Mass Spectrometry. J Chromatograph Separat Techniq S6:003. doi:10.4172/2157-7064.S6-003
Page 2 of 6
Special Issue 5 • 2015
J Chromatograph Separat Techniq
ISSN:2157-7064 JCGST, an open access journal
For example, 13C, atom percent 13C=[13C/(12C+13C)] × 100
Various applications of the isotopic abundance study includes (a)
the distribution of contaminant sources of any molecule on a native,
regional, and global scale, (b) the identication and quantication of
alteration reactions, and (c) the characterization of elementary reaction
mechanisms that govern product formation [15].
Both of the phenol derivatives taken for this study, work via the
formation of free radicals. Free radicals are highly unstable species.
Due to their highly oxidative characteristics, these free radicals may
contribute to carcinogenicity or tumorigenicity; however the same
reactions may combat oxidative stress. Hence, the stability of phenol
derivatives is important to show enhanced antioxidant properties. is
could be enhanced by Mr. Trivedi’s unique bioeld energy treatment
which is already known to alter the physical and structural properties
of various living and non-living substances [16]. e National Center
for Complementary and Alternative Medicine (NCCAM) which is part
of the National Institute of Health (NIH), has recommended the use
of Complementary and Alternative Medicine CAM therapies in the
healthcare sector and about 36% of Americans regularly use some form
of CAM [17]. CAM includes numerous energy-healing therapies, in
which bioeld therapy is a form of putative energy medicine that is being
widely used worldwide to improve the overall health and well-being of
human beings. When an electrical signal passes through any material, a
magnetic eld is generated in the surrounding space [18]. Humans have
the ability to harness energy from the environment/universe and can
then transmit in to any object (living or non-living) around the globe.
e object(s) always receive the energy and respond in a useful way.
is process is called bioeld energy treatment. Mr. Trivedi’s unique
bioeld energy treatment is also called e Trivedi Eect®, which is
known to alter the physical, structural and atomic properties in various
metals [19-21] and ceramics [22,23] in materials science. Additionally,
the impact of bioeld treatment has been studied in various elds
like microbiology research [16,24], biotechnology research [25,26],
and agriculture research [27,28]. Our group of scientists reported
that the bioeld treatment substantially altered the atomic, structural
and physical properties in silicon carbides [29] and carbon allotropes
[30]. Based on the outstanding results achieved by bioeld treatments
on metals and ceramics, an attempt was made to evaluate the eect of
bioeld treatment on the isotopic abundance ratio of 13C/12C or 2H/1H
(PM+1)/PM and 18O/16O (PM+2)/PM in BHT and 4-MP.
Experimental
Both BHT and 4-MP were procured from SD Fine Chem. Ltd.,
India. Each of the samples i.e., BHT and 4-MP were distributed into
two parts, where one part of each sample was referred as control sample
and the other part was considered as treatment group. e treatment
group was handed over to Mr. Trivedi for bioeld treatment in sealed
pack and under standard laboratory conditions. Mr. Trivedi provided
the treatment through his energy transmission process to the treated
group without touching the sample. e control and treated samples
were characterized using Gas Chromatography-Mass Spectrometry
(GC-MS).
GC-MS method
e GC/MS was performed in a silica capillary column. It is
equipped with a quadrupole detector with pre-lter, one of the fastest,
widest mass ranges available for any GC/MS. e mass spectrometer
was operated in an electron ionization (EI) positive/negative, and
chemical ionization mode at the electron ionization energy of 70 eV.
Mass range: 20-620 Daltons (amu), stability: ± 0.1 m/z mass accuracy
over 48 hours. e identication of analytes were done by retention
time and by a comparison of the mass spectra of identied substances
with references. 2 µL was injected in splitless mode. e carry gas (N2)
ow-rate was kept constant during the run at 2 ml min-1. e stock
standard solution of BHT and 4-MP were prepared in methanol (HPLC
grade) to a concentration of 100 µg/mL. Working standard solutions
were prepared from the stock solutions.
Both the control and treated samples were injected following the
same experimental protocol. For GC-MS analysis the treated sample
was further divided into three parts as T1, T2, T3 and T4. e GC-MS
data was obtained in the form of % abundance vs. mass to charge ratio
(m/z), which is known as mass spectrum. e isotopic abundance ratio
13C/12C or 2H/1H (PM+1)/PM and 18O/16O (PM+2)/PM was expressed
by its deviation in treated samples as compared to the control. e
percentage change in isotopic ratio (PM+1/PM) and (PM+2/PM) were
calculated on a percentage scale from the following formula:
RR
treated control
Percent change in isotopic abundance ratio (PM 1/ PM) 100
Rcontrol
−
+= ×
Where, RTreat ed and RControl are the ratio of intensity at (PM+2)
and (PM+1) to PM in mass spectra of treated and control samples
respectively.
Solution preparation
Compounds were dissolved in methanol (HPLC grade) and ltered
with 0.2 micron lter tip tted with a syringe. Phenol derivatives were
polar molecule and, therefore, a polar solvent methanol was used as
the diluent. Minimum detection limit of the instrument is up to 1
picogram masses. Working standard solutions were prepared from the
stock solutions (concentration 100 µg/mL). e spectra was obtained
by injecting 2 µL standard solution of the phenol components in
methanol. A solvent delay of ~4 minutes eliminates the appearance of
the methanol solvent peak in this chromatogram.
Results and Discussion
GC-MS spectroscopy
GC-MS spectra of BHT: e GC-MS spectra of control, treated
samples (T1, T2), and (T3, T4) are presented in Figures 1, 2 and 3,
respectively. MS spectra showed PM peak at m/z = 220 and base peak
at m/z = 205 in all control and treated BHT samples (T1, T2, T3 and
T4). e intensity ratio of PM+1 (i.e., m/z=221) and PM (i.e., m/z=220)
peaks are presented in Table 1. Eight major peaks at m/z= 220, 205,
177, 145, 105, 91, 57, and 41 were observed in control sample due to
degradation of BHT, corresponded to the following ions respectively:
C15H24O+, C14H21O+, C12H17O+, C10H9O+, C8H9
+, C7H7
+, C4H9
+, and
C3H5
+. Peak at m/z=205 was observed due to C14H21O+ and a methyl
cation from BHT. While peaks at m/z= 41 and 177 were observed due
to the fragmentation of tertiary butyl group of BHT to propene and
C12H17O+, respectively. Peaks at m/z=145 and 57 were observed due to
the formation of an m-xylene derivative and butane. Finally m-xylene
was observed at m/z=105 in BHT fragmentation. More suggestively
the treated BHT samples (T1-T4) were fragmented in the same way
as compared to control and showed eight major peaks in the mass
spectrum with dierent intensities. All peaks (m/z =220, 205, 177, 145,
105, 91, 57, and 41) were same for both treated and control samples
[31].
However, a signicant alteration in isotopic abundance ratio of
(PM+1)/PM and (PM+2)/PM was observed for treated samples of
BHT as compared to control. e isotopic abundance ratio in control
Citation: Trivedi MK, Branton A, Trivedi D, Nayak G, Saikia G, et al. (2015) Investigation of Isotopic Abundance Ratio of Bioeld Treated Phenol
Derivatives Using Gas Chromatography-Mass Spectrometry. J Chromatograph Separat Techniq S6:003. doi:10.4172/2157-7064.S6-003
Page 3 of 6
Special Issue 5 • 2015
J Chromatograph Separat Techniq
ISSN:2157-7064 JCGST, an open access journal
Figure 1: GC-MS spectrum of control butylatedhydroxytoluene sample.
Figure 2: GC-MS spectra of treated butylatedhydroxytoluene sample T1 and T2.
Figure 3: GC-MS spectra of treated butylatedhydroxytoluene sample T3 and
T4.
0.58 1.83
38.39
181.27
0
50
100
150
200
T1 T2 T3 T4
Percent change
Figure 4: Percent change in isotopic abundance (PM+1/PM) of
butylatedhydroxytoluene under bioeld treatment as compared to control.
and treated BHT was calculated and presented in the Figures 4 and
5 respectively. e isotopic abundance ratio of (PM+1)/PM of BHT
treated sample was increased exponentially from T1 to T4 (T1=0.58%,
T2=1.83%, T3=38.39%, and T4=181.27%) as compared to control. In
case of (PM+2) isotope same trend was followed, isotopic abundance
ratio of (PM+2)/PM was slightly decreased by 0.65% for, T1 sample
and then increased up to 185.99% in case of T4 (T1=-0.65%, T2=2.81%,
T3=38.34% and T4=185.99%). e increased isotopic abundance ratio
of (PM+1)/PM and (PM+2)/PM in treated BHT may increase eective
mass (µ) and binding energy in this molecules with heavier isotopes.
GC-MS spectra of 4-MP: PM peak was observed at m/z=124 in
both control and treated samples with dierent intensity ratio (Figures
-0.65 2 .81
38.34
185.99
-50
0
50
100
150
200
T1 T2 T3 T4
Percent change
Figure 5: Percent change in isotopic abundance (PM+2/PM) of
butylatedhydroxytoluene under bioeld treatment as compared to control.
Citation: Trivedi MK, Branton A, Trivedi D, Nayak G, Saikia G, et al. (2015) Investigation of Isotopic Abundance Ratio of Bioeld Treated Phenol
Derivatives Using Gas Chromatography-Mass Spectrometry. J Chromatograph Separat Techniq S6:003. doi:10.4172/2157-7064.S6-003
Page 4 of 6
Special Issue 5 • 2015
J Chromatograph Separat Techniq
ISSN:2157-7064 JCGST, an open access journal
Figure 6: GC-MS spectrum of control sample of 4-methoxyphenol.
Figure 8: GC-MS spectra of treated samples of 4-methoxyphenol (T3 and T4).
-8.07
380.73
104.28 103.9
-100
0
100
200
300
400
500
T1 T2 T3 T4
Percent change
Figure 9: Percent change in isotopic abundance (PM+1/PM) of 4-methoxyphenol
under bioeld treatment as compared to control.
Figure 7: GC-MS spectra of treated samples of 4-methoxyphenol (T1 and T2).
-10.16
355.33
96.57 102.27
-100
0
100
200
300
400
T1 T2 T3 T4
Percent change
Figure 10: Percent change in isotopic abundance (PM+2/PM) of 4-methoxyphenol
under bioeld treatment as compared to control.
Citation: Trivedi MK, Branton A, Trivedi D, Nayak G, Saikia G, et al. (2015) Investigation of Isotopic Abundance Ratio of Bioeld Treated Phenol
Derivatives Using Gas Chromatography-Mass Spectrometry. J Chromatograph Separat Techniq S6:003. doi:10.4172/2157-7064.S6-003
Page 5 of 6
Special Issue 5 • 2015
J Chromatograph Separat Techniq
ISSN:2157-7064 JCGST, an open access journal
6-8). e intensity ratio of PM peak and (PM+1) peak are given in Table
2. Total ve major peaks at m/z=124, 109, 81, 53, and 39 were observed
for both control and treated samples of 4-MP due to C7H8O2
+
, C6H5O2
+,
C6H9
+
, C4H5
+ and C3H3
+ ions, respectively. Peak at m/z=109 and 81 were
seen due to the initial fragmentation of 4-MP to hydroquinone (base
peak) and methyl radical where hydroquinone was further reduced
to cyclohexene (m/z=81). Peaks at 53 and 39 was observed due to the
formation of 1,3-butadiene and propa-1,2-diene from the ring opening
reaction of 4-MP. One low intensity peak at m/z=27 was observed in
all treated (T1-T4) 4-MP samples which was due to the formation of
ethene ion (C2H3
+). Fragmentation pattern of both control and treated
4-MP molecules were same, however number of fragmented peaks
were increased from control to treated samples [32].
Isotopic abundance ratio of (PM+1)/PM and (PM+2)/PM of
control and treated 4-MP was calculated and presented in the Figures
9 and 10, respectively. e isotopic abundance ratio of (PM+1)/PM of
treated 4-MP was increased signicantly up to 380.73% (T1=-8.07%,
T2=380.75% T3=104.28%, and T4=103.9%) under bioeld treatment.
e isotopic abundance ratio of (PM+2)/PM was also increased in a
similar way up to 355.33% (T1=-10.16%, T2=355.33% T3=96.57%, and
T4=102.27%).
Atoms taking part in chemical bonds with higher isotopic number
might have higher binding energy with increased eective mass (µ)
and vice versa. us, the increased isotopic abundance ratio of 13C/12C
or 2H/1H and 18O/16O in both the compounds (BHT and 4-MP) might
increase the eective mass and binding energy aer bioeld treatment
that may enhance the stability of phenol derivatives signicantly. e
increased isotopic abundance ratio of (PM+1)/PM and (PM+2)/PM in
samples of BHT and 4-MP aer bioeld treatment, may result in chemical
stability. It could be due to nuclear level transformation of (PM+1) and
PM, which may induced through bioeld treatment. e observed
fragmentation pattern and number fragmented peaks were same for
control and treated samples of BHT and while number fragmented
peaks in treated samples of 4-MP were increased. e increased
isotopic abundance ratio may reasonably increase the no of heavier
isotopes (i.e., 2H, 13C and 18O) in the molecule aer bioeld treatment.
If a lighter nucleus is replaced by a heavier one then corresponding
eective mass (µ) of that particular bond could be changed accordingly.
We have presented some probable bonds that might present such as
12C-12C, 1H-12C, 13C-12C, 2H-12C, 1H-13C, 2H-13C,13C-13C, 12C-18O, 1H-18O,
13C-16O, and 2H-16O in the bioeld treated molecules. Eective mass is
calculated and presented in Table 3. e result showed that µ of normal
12C-12C and 1H-12C bond was 6 and 0.923, respectively. It showed that
reduced mass is increased in case of heavier isotope (i.e., 12C-13C=6.26,
and, 2H-12C=1.71). e increased µ was observed in case of 18O-12C and
1H-18O bonds also. From the Figures 4, 5, 9 and 10, it was observed
that the isotopic abundance ratio of (PM+1)/PM in treated samples
increased by 181.27% and 380.73% in BHT and 4-MP. So maximum
number of isotope replacement may occur in this process, which may
lead to signicant change in energy of the isotope substituted bonds. It
may enhance the bond strength, stability, and binding energy of BHT
and 4-MP molecules.
Conclusions
In summary, BHT and 4-MP were studied under the inuence of
bioeld energy treatment and observed signicant changes in isotopic
abundance compared to the control sample. e bioeld treatment
oers a remarkable means to alter the isotopic abundance ratio of
13C/12C or 2H/1H (PM+1/PM) and 18O/16O (PM+2/PM) in BHT, as well
as 4-MP, which have a signicant impact on bond energies and the
chemical reactivity of the molecules. e percent change in the isotopic
abundance ratio of (PM+1)/PM and (PM+2)/PM in the treated BHT
was increased up to 181.27% and 185.99% respectively as compared
to the control. However, the percent change in the isotopic abundance
ratio of (PM+1)/PM and (PM+2)/PM in treated 4-MP was increased
up to 380.73% and 355.33%, respectively. GC-MS data suggests that
bioeld treatment has signicantly increased the isotopic abundance of
2H, 13C and 18O in treated BHT and 4-MP. Stability may be increased
by increasing the eective mass (µ), which consequently increases the
binding energy. e increased isotopic abundance ratio aer bioeld
treatment on BHT and 4-MP molecules may increase the bond stability,
which could result in reduce autocatalytic oxidation reactions and thus
prevent ageing and most common reactions initiated by heat, light and
molecular oxygen.
Acknowledgments
The authors would like to acknowledge the whole team from the Sophisticated
Analytical Instrument Facility (SAIF), Nagpur for providing the instrumental facility.
We are very grateful for the support from Trivedi Science, Trivedi Master Wellness
and Trivedi Testimonials in this research work.
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Parameters Control Treated
T1 T2 T3 T4
Peak Intensity at m/z=(PM) 56.32 57.46 66.62 77.57 84.36
Peak Intensity at m/z=(PM+1) 8.41 8.63 10.13 16.03 35.75
Peak Intensity at m/z=(PM+2) 0.74 0.75 0.90 1.41 3.17
Table 1: GC-MS isotopic abundance analysis result of butylatedhydroxytoluene.
Parameters Control Treated
T1 T2 T3 T4
Peak Intensity at m/z=(PM) 94.46 92.36 90.27 100 99.71
Peak Intensity at m/z=(PM+1) 7.81 7.02 35.88 16.89 16.81
Peak Intensity at m/z=(PM+2) 0.74 0.65 3.22 1.54 1.58
Table 2: GC-MS isotopic abundance analysis result of 4-methoxyphenol.
Isotopes Bonds Isotope type Reduced mass [mAmB /(mA+mB)]
12C-12C Lighter 6.00
13C-12C Heavier 6.26
1H-12C Lighter 0.923
1H-13C Heavier 0.929
2H-12C Heavier 1.71
1H-18O Heavier 0.94
2H-16O Heavier 1.77
18O-12C Heavier 7.20
16O-13C Heavier 7.17
Table 3: Possible isotopic bonds in butylatedhydroxytoluene and 4-methoxyphenol.
mA: Mass of an atom A; mB: Mass of an atom B; A may be C or H and so on.
Citation: Trivedi MK, Branton A, Trivedi D, Nayak G, Saikia G, et al. (2015) Investigation of Isotopic Abundance Ratio of Bioeld Treated Phenol
Derivatives Using Gas Chromatography-Mass Spectrometry. J Chromatograph Separat Techniq S6:003. doi:10.4172/2157-7064.S6-003
Page 6 of 6
Special Issue 5 • 2015
J Chromatograph Separat Techniq
ISSN:2157-7064 JCGST, an open access journal
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