Research Article
Isotopic Abundance Analysis Ratio of Consciousness Energy Healing Treated 6-Mercaptopurine Using LC-MS and GC-MS Spectrometry
Alice Branton, Mahendra Kumar Trivedi, Dahryn Trivedi, Snehasis Jana*
Corresponding Author: Snehasis Jana, Trivedi Science Research Laboratory Pvt. Ltd., Thane-West, Maharashtra, India
Received: November 4, 2020; Revised: January 10, 2021; Accepted: December 29, 2020 Available Online: January 26, 2021
Citation: Branton A, Trivedi MK & Trivedi D, Jana S. (2021) Isotopic Abundance Analysis Ratio of Consciousness Energy Healing Treated 6-Mercaptopurine Using LC-MS and GC-MS Spectrometry. J Drug Design Discov Res, 2(1): 58-66.
Copyrights: ©2020 Jana S, Branton A, Trivedi MK & Trivedi D. 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.
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The 6-Mercaptopurine, an antineoplastic agent, is useful for the treatment of many diseases. This study was performed to investigate the impact of The Trivedi Effect®-Biofield Energy Healing Treatment on the structural properties and the isotopic abundance ratio of 6-mercaptopurine using liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) spectroscopy. The 6-mercaptopurine sample was divided into two parts - one part was considered as a control sample (i.e., no Biofield Energy Treatment was provided), while the other part was considered as a treated sample (i.e., treated with The Trivedi Effect®-Consciousness Energy Healing Treatment remotely by a renowned Biofield Energy Healer, Alice Branton). The LC-MS spectra of both the samples at retention time (Rt) 2.24 minutes exhibited the mass of the protonated molecular ion peak at m/z 153 [M+H] + (calculated for C5H5N4S+, 153.18). The LC-MS based isotopic abundance ratio of PM+1/PM (2H/1H or 13C/12C or 15N/14N or 33S/32S) in the treated 6-mercaptopurine was significantly decreased by 42.49% compared with the control sample. Thus, 13C, 2H, 15N, and 33S contributions from (C5H5N4S) + to m/z 154 in the treated sample were significantly decreased compared with the control sample. The GC-MS based isotopic abundance ratio of PM+1/PM in the treated 6-mercaptopurine was decreased by 1.34% compared with the control sample. Hence, 13C, 2H, 15N, and 33S contributions from (C5H5N4S)+ to m/z 153 in the treated sample were significantly decreased compared with the control sample. However, the isotopic abundance ratio of PM+2/PM in the treated 6-mercaptopurine was significantly increased by 11.58% compared with the control sample. Therefore, 18O contributions from (C5H5N4S) + to m/z 154 in the treated sample were significantly increased compared with the control sample. The isotopic abundance ratio of PM+1/PM (2H/1H or 13C/12C or 15N/14N or 33S/32S) and PM+2/PM (34S/32S) in the treated 6-mercaptopurine was significantly altered compared to the control sample. It can be assumed that the changes in isotopic abundance and mass peak intensities could be due to changes in nuclei possibly through the interference of neutrino particles via The Trivedi Effect® - Consciousness Energy Healing Treatment. The new form of treated 6-mercaptopurine would be better in designing novel pharmaceutical formulations that might offer a better therapeutic response against acute lymphocytic leukemia, chronic myeloid leukemia, Crohn's disease, ulcerative colitis, etc.

Keywords: 6-Mercaptopurine, The Trivedi Effect®, Biofield Energy, Consciousness Energy Healing Treatment, LC-MS, GC-MS

INTRODUCTION

The 6-mercaptopurine is an antimetabolite anticancer agent. It acts by interfering with nucleic acid synthesis by inhibiting purine metabolism [1,2]. It is used for the medication of cancer and autoimmune diseases, i.e., acute lymphocytic leukemia, chronic myeloid leukemia, Crohn's disease, and ulcerative colitis [3-5]. It has an immunosuppressant property. It was approved for medical use in the U.S.A. since 1953 and also listed as Essential Medicines by the World Health Organization (WHO) [6]. Very common side effects like diarrhea, nausea, vomiting, loss of appetite, stomach and abdominal pain, mouth sores, fatigue, weakness, fever, sore throat, skin rash, pinpoint red spots on the skin, darkening of the skin, yellowing of eyes or skin, hair loss, easy bruising or bleeding, bloody stools, black or tarry stools, dark urine, bloody urine, painful or difficult urination, may suppress the production of blood cells (both white and red blood cells) and can be linked to the genetic polymorphisms are associated with the mercaptopurine therapy [7-9]. Mercaptopurine is used in the pharmaceutical formulations for oral medications in the form of the tablet and liquid suspension [10-12]. The solubility profile of mercaptopurine is very poor, which is insoluble in water, acetone, chloroform, and diethyl ether, whereas slightly soluble in dilute sulfuric acid; soluble in hot alcohol and dilute alkali solutions [12].

Improvement of the physicochemical properties is very important for the improvement of solubility, dissolution, absorption, and bioavailability of the pharmaceutical and nutraceutical compounds [13]. In this regard, The Trivedi Effect®-Biofield Energy Healing Treatment has a significant effect on various properties such as particle size, surface area, bioavailability and isotopic abundance ratios of pharmaceutical and nutraceutical compounds [14-18]. The Trivedi Effect® is a natural and only scientifically proven phenomenon in which a person can harness this inherently intelligent energy and transmit it anywhere on the planet through the possible mediation of neutrinos [19]. “Biofield Energy” the electromagnetic energy field which exists surrounding the living beings, which can transmit the electromagnetic energy in the form of bio-photons, generated by the continuous movement of the electrically charged particles (ions, cells, etc.) inside the body. Biofield Energy Healing specialists can harness the energy from the environment or the “Universal Energy Field” and can transmit into any living and non-living object(s), this process is called Biofield Energy Healing Treatment [20-22]. Biofield based Energy Therapies have been reported with significant outcomes against various disease [23]. 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 yoga, Qi Gong, Tai Chi, hypnotherapy, Reiki, etc. [24,25]. These therapies have been accepted by most of the U.S.A. population with several advantages [25]. The Trivedi Effect®-Consciousness Energy Healing Treatment has been reported with the significant revolution in the metals, chemicals, ceramics and polymers, crops, microbes, biotechnology, skin health, bone health, cancer cell line, etc. [24-38]. The Trivedi Effect®-Biofield Energy Healing Treatment could be an economical approach for the practical problems associated with 6-mercaptopurine for the physicochemical properties for designing better pharmaceutical formulations. The stable isotope ratio analysis has various applications in different scientific fields for understanding the isotope effects resulting from the variation of the isotopic composition of the molecule [39,40]. Isotope ratio analysis can be performed by using the conventional mass spectrometry (MS) techniques such as gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) in low micromolar concentration with sufficient precision [39,41]. Therefore, LC-MS and GC-MS were used in this study to characterize the structural properties and evaluate the isotopic abundance ratio analysis of PM+1/PM (2H/1H or 13C/12C or 15N/14N or 33S/32S) and PM+2/PM (33S/32S) in The Trivedi Effect® - Consciousness Energy Healing treated 6-mercaptopurine compared to the control sample.

MATERIALS AND METHODS

Chemicals and reagents

6-mercaptopurine was purchased from Tokyo Chemical Industry Co., Ltd., Japan. Other chemicals used during the experiments were of analytical grade available in India.

Consciousness Energy Healing Treatment Strategies

The 6-mercaptopurine powder was the test sample divided into two parts. One part of the 6-mercaptopurine powder sample was considered as a control sample (no Biofield Energy Treatment was provided). However, the other part of 6-mercaptopurine was treated with The Trivedi Effect®- Consciousness Energy Healing Treatment remotely under standard laboratory conditions for 3 minutes and known as The Trivedi Effect® Treated or Biofield Energy Treated 6-mercaptopurine sample. The Biofield Energy Treatment was provided through the healer’s unique energy transmission process by the renowned Biofield Energy Healer, Alice Branton, USA, to the test sample. Further, the control sample was treated with a “sham” healer for comparison purposes. The sham healer did not have any knowledge about the Biofield Energy Treatment. After that, the Biofield Energy Treated and untreated 6-mercaptopurine samples were kept in sealed conditions and characterized using LC-MS and GC-MS, analytical 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 6-mercaptopurine was carried out with the help of LC-MS Thermo Fisher Scientific, the USA equipped with an ion trap detector connected with a triple-stage quadrupole mass spectrometer. The column used here was a reversed-phase Thermo Scientific Synchronis C18 (Length-250 mm X ID 4.6 mm X 5 micron), maintained at 25˚C. The diluent used for the sample preparation was acetonitrile and water. 10 µL of the 6-mercaptopurine solution was injected, and the analyte was eluted using 0.1% formic acid in water (mobile phase A; 10%) and acetonitrile (mobile phase B; 95%) pumped at a constant flow rate of 0.5 mL/min. Chromatographic separation was achieved using gradient condition and the total run time was 10 min. Peaks were monitored at 300 nm using the Photo Diode Array (PDA) detector. The mass spectrometric analysis was performed under a +ve ESI mode. The total ion chromatogram, peak area% and mass spectrum of the individual peak which was appeared in LC along with the full scan (m/z 50-300) were recorded. The total ion chromatogram and mass spectrum of the individual peak (appeared in LC-MS) were recorded.

The natural abundance of each isotope (C, O, H, N, and S) can be predicted from the comparison of the height 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 [40,41-44]. The LC-MS based isotopic abundance ratios (PM+1/PM) for the control and Biofield Energy Treated 6-mercaptopurine was calculated.

Percentage (%) change in isotopic abundance ratio = [(IARTreated – IARControl)/ IARControl) x 100]
where IARTreated = isotopic abundance ratio in the treated sample and IARControl = isotopic abundance ratio in the control sample.

Gas chromatography-mass spectrometry (GC-MS) analysis

GC-MS of the control and Biofield Energy Treated sample of 6-mercaptopurine were analyzed with the help of Perkin Elmer Gas chromatograph equipped with a PE-5MS (30 M x 250 microns x 0.250 microns) capillary column and coupled to a single quadrupole mass detector was operated with electron impact (EI) ionization in positive mode. The oven temperature was programmed from 180°C (14 min hold) to 290°C (14 min hold) @ 10°C/min (total run time 30 min). The sample was prepared taking 80 mg of the 6-mercaptopurine is in 4 ml acetonitrile and water (1:1) as a diluent. Mass spectra were scanned from m/z 20 to 400. The identification of analyte was done by GC retention times and by a comparison of the mass spectra of samples.

The GC-MS based isotopic abundance ratios (PM+1/PM and PM+2/PM) for the control and Biofield Energy Treated 6-mercaptopurine was calculated.

Percentage (%) change in isotopic abundance ratio = [(IARTreated – IARControl)/ IARControl) x 100]
Where IARTreated = isotopic abundance ratio in the treated sample and IARControl = isotopic abundance ratio in the control sample.

RESULTS AND DISCUSSION

Liquid chromatography-mass spectrometry (LC-MS)

The LC-MS chromatograms and mass spectra of both the samples of 6-mercaptopurine are shown in Figures 1 and 2, respectively. The chromatograms of 6-mercaptopurine showed the single major chromatographic peak at the retention time (Rt) of 2.24 minutes in the case of both the samples (Figure 1). These results indicated that the polarity of both the control and Biofield Energy Treated 6-mercaptopurine was the same. As per the literature 6-mercaptopurine was detected with the molecular mass peak [M]+ at m/z 152 MS spectrum in positive ion mode [12]. The mass spectra of both the samples of 6-mercaptopurine (Figure 2) exhibited the mass of the protonated molecular ion peak at m/z 153 [M+H] + (calculated for C5H5N4S+, 153.18) in the control sample and Biofield Energy Treated sample (Figure 3).






The LC-MS spectra of both the control and Biofield Energy Treated 6-mercaptopurine showed the mass of the molecular ion peak [M+H] + at m/z 153 [M+H] + (calculated for C5H5N4S+, 153.18) with the relative intensity of 100%. The theoretical calculation of PM+1 for 6-mercaptopurine was presented below:
P (13C) = [(5 x 1.1%) x 100% (the actual size of the M+ peak)]/100% = 5.5%
P (2H) = [(5 x 0.015%) x 100%] / 100%= 0.075%
P (15N) = [(4 x 0.4%) x 100%] / 100% = 1.6%
P (33S) = [(1 x 0.08%) x 100%] / 100% = 0.08%
PM+1, i.e., 13C, 2H, 15N, and 33S contributions from (C5H5N4S) + to m/z 154= 7.26%
The calculated isotope abundance (7.26%) was close to the experimental value of 8.19% (Table 1). From the above calculation, it has been found that 13C and 15N have a major contribution to m/z 154.
The LC-MS based isotopic abundance ratio analysis PM and PM+1 for 6-mercaptopurine near m/z 153 and 154, respectively of the control and Biofield Energy Treated samples, which were obtained from the observed relative peak intensities of [M+] and [(M+1) +] peaks, respectively in the ESI-MS spectra (Table 1). The percentage change of the isotopic abundance ratio (PM+1/PM) in the Biofield Energy Treated 6-mercaptopurine was significantly decreased by 42.49% compared with the control sample (Table 1). Therefore, it was concluded that the 13C, 2H, 15N, and 33S contributions from (C5H5N4S) + to m/z 154 in the Biofield Energy Treated sample were significantly decreased compared to the control sample.

Gas chromatography-mass spectrometry (GC-MS) analysis

The GC-MS of the control and Biofield Energy Treated 6-mercaptopurine showed the presence of the single chromatographic peak at the retention time of 16.04 min in the chromatogram (Figures 4 and 5). The parent molecular ion peak of 6-mercaptopurine at m/z 152 [M]+ (calculated for C5H5N4S+, 152.02) in the control sample and Biofield Energy Treated sample, along with the fragment ion peaks near m/z 119 and 70 (Figures 4 and 5) which were proposed corresponded to the molecular formula C5H3N4+ and C3H6N2+, respectively (Figure 3). The mass peak intensities influence the isotopic abundance ratio, which was well supported by the LC-MS based isotopic abundance ratio analysis.


The GC-MS spectra of both the control and Biofield Energy Treated 6-mercaptopurine showed the mass of the molecular ion peak [M]+ at m/z 152 (calculated for C5H4N4S+, 152.02). The theoretical calculation of PM+1 for 6-mercaptopurine was presented as below:
P (13C) = [(5 x 1.1%) x 100% (the actual size of the M+ peak)] / 100% = 5.5%
P (2H) = [(4 x 0.015%) x 100%] / 100%= 0.06%
P (15N) = [(4 x 0.4%) x 100%] / 100% = 1.6%
P (33S) = [(1 x 0.08%) x 100%] / 100% = 0.08%
PM+1, i.e., 13C, 2H, 15N, and 33S contributions from (C5H5N4S) + to m/z 153 = 7.24%
From the above calculation, it has been found that 13C and 15N have a major contribution to m/z 153.
Similarly, the theoretical calculation of PM+2 for 6-mercaptopurine was presented as below:
P (34S) = [(1 x 4.21%) x 100%] / 100% = 4.21%
PM+2, i.e., 34S contributions from (C5H5N4S) + to m/z 154 = 4.21%
From the above calculation, it has been found that only 34S has a major contribution to m/z 153. The calculated isotopic abundances (4.21) were close to the experimental value of 3.8 (Table 2).
The GC-MS based isotopic abundance ratio analysis of 6-mercaptopurine in the control and treated samples were calculated. PM, PM+1, and PM+2 for 6-mercaptopurine near m/z 152, 153, and 154, respectively of the control and treated samples, which were obtained from the observed relative peak intensities of [M+], [(M+1) +], and [(M+2) +] peaks, respectively in the mass spectra and are presented in Table 2. The isotopic abundance ratio of PM+1/PM in the Biofield Energy Treated 6-mercaptopurine was decreased by 1.34% compared with the control sample (Table 2). Hence, 13C, 2H, 15N, and 33S contributions from (C5H5N4S) + to m/z 153 in the treated sample were decreased compared with the control sample. However, the isotopic abundance ratio of PM+2/PM in the treated was significantly increased by 11.58% compared with the control sample (Table 2). Hence, 34S contributions from (C5H5N4S) + to m/z 154 in the treated sample were significantly increased compared with the control sample.

LC-MS and GC-MS study confirmed the structure of the sample as 6-mercaptopurine. The isotopic abundance ratios of PM+1/PM (2H/1H or 13C/12C or 15N/14N or 33S/32S) and PM+2/PM (34S/32S) in the treated sample were significantly altered compared to the control sample. As per modern physics, neutrinos change identities which are only possible if the neutrinos possess mass and could interchange their phase from one phase to another internally. Therefore, the neutrinos could interact with protons and neutrons in the nucleus, which indicated a close relation between neutrino and the isotope formation [19,40,41]. The altered isotopic composition at the molecular level of The Trivedi Effect®-Consciousness Energy Healing Treated 6-mercaptopurine might be due to the alteration in neutron to proton ratio in the nucleus. It can be hypothesized that the changes in isotopic abundance could be due to changes in nuclei possibly through the interference of neutrino particles via The Trivedi Effect® - Consciousness Energy Healing Treatment. The new form of 6-mercaptopurine (Biofield Energy Treated) would be particularly useful to design better pharmaceutical formulations that might offer a better therapeutic response against many diseases.

CONCLUSIONS
The experimental results concluded that The Trivedi Effect®-Consciousness Energy Healing Treatment showed a significant impact on the isotopic abundance ratios and mass peak intensities of 6-mercaptopurine. The LC-MS based isotopic abundance ratio of PM+1/PM (2H/1H or 13C/12C or 15N/14N or 33S/32S) in the Biofield Energy Treated 6-mercaptopurine was significantly decreased compared with the control sample. Thus, 13C, 2H, 15N, and 33S contributions from (C5H5N4S) + in the Biofield Energy Treated sample were significantly decreased compared with the control sample. The GC-MS based isotopic abundance ratio of PM+1/PM in the Biofield Energy Treated 6-mercaptopurine was decreased as compared with the control sample. Hence, 13C, 2H, 15N, and 33S contributions from (C5H5N4S) + in the Biofield Energy Treated sample were significantly decreased compared with the control sample. However, the isotopic abundance ratio of PM+2/PM in the Biofield Energy Treated 6-mercaptopurine was significantly increased compared with the control sample. Hence, 18O contributions from (C5H5N4S) + in the Biofield Energy Treated sample were significantly increased compared with the control sample. It can be assumed that the changes in isotopic abundance and mass peak intensities could be due to changes in nuclei possibly through the interference of neutrino particles via The Trivedi Effect® - Consciousness Energy Healing Treatment (Biofield Energy Healing Treatment). The new form of Biofield Energy Treated 6-mercaptopurine would be better designing novel pharmaceutical formulations that might offer a better therapeutic response against acute lymphocytic leukemia, chronic myeloid leukemia, Crohn's disease, and ulcerative colitis, etc.

ACKNOWLEDGMENTS

The authors are grateful to the Sophisticated Instrumentation Centre for Applied Research & Testing (SICART) India, Trivedi Science, Trivedi Global, Inc., Trivedi Testimonials, and Trivedi Master Wellness for their assistance and support during this work.
 
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