INTRODUCTION:

For many years spin traps have been used to increase the stability of free radicals in order for them to be identified and detected by electron paramagnetic resonance (EPR) spectroscopy. Spin traps are highly reactive toward free radicals, thereby allowing the acquisition of abundant information on the production of such species in biological, biochemical, and chemical systems. Spin traps have been used extensively in the detection of oxygen radicals^1-4 in order to detect and quantify the involved radical species. One of the most reactive oxygen radical was hydroxyl radical and it is consequently shot-term lived known radical⁸. Hydroxyl radicals damages macro-molecules like DNA^5-6. Within the cell, the hydroxyl radicals can upset the redox balance which directs to the activation of transcription factors such as NF-kB or AP-I⁹.

Spin trapping entails the reaction of nitrones or nitroso spin traps (paramagnetic species) with unstable free radical systems, in order to form a more stable free radical (radical adduct), which can be usually detected by EPR spectroscopy⁷. But this method was limited due to the weakness of the derived nitroxides to reduction by cellular reductants like Ascorbate, the result formed was hydroxylamine which was undetectable by using EPR.

And one more limitation of this EPR technique was it makes difficult to differentiate between the similar spectra which are the yields of many radical adducts, having different structures. These limitations are important for the need of improved methods of radical-adduct detection, structural determination and quantification. In the recent years there has been a major importance for the technique was the combination of Liquid Chromatography Electron Paramagnetic Resonance [LC/EPR] with online Liquid Chromatography/Mass Spectrometry [LC/MS]. Many ESR spin trapping studies also use GC/MS for the structural characterization once the radical adduct has been identified^10-11.

In the present study, the combination of gas chromatography/mass spectrometry for the detection of trapped radical species has never been used previously and Proxyl derivatives are used as spin trapping agents and hydroxyl radicals are generated by using the Fenton reaction¹² in which an EDTA complex reacting with H₂O₂ in the presence of ascorbic acid. Here in this study we have used an important reaction where a hydroxyl radical reacts with dimethyl sulfoxide and ethanol converting it to methyl and ethyl radicals. With this technique we detect hydroxyl radicals indirectly by trapping methyl and ethyl radicals.

MATERIAL AND METHODS:

3-carboxy-Proxyl, 3-carbamoyl-Proxyl was kindly gifted by M/s Natco Fine Pharmaceuticals, Hyderabad. Ascorbic acid, EDTA, DMSO, Dipotassium hydrogen phosphate (K₂HPO₄), and ferrous ammonium sulphate (Fe (NH₄)₂(SO₄)₂.6H₂O) were purchased from s. d. Fine Chemicals Pvt. Ltd., Mumbai. And the other reagents like hydrogen peroxide, chloroform were obtained from commercial sources. All chemicals used were of analytical grade.

1. Standard reaction conditions:

The process which is used in this method is standard and should be followed throughout the experiment. First, 100mM of potassium phosphate buffer (5ml) was prepared and adjusted its pH to 7.4 by using concentrated hydrochloric acid where the in-vitro interactions of DMSO and OH willoccur. Next the following prepared solutions are added in a strict order:

a. 11mM ethylene di-amine tetra acetic acid (EDTA) solution (1ml),

b. 50mMspin trap solution (1ml),

c. 3% hydrogen peroxide solution (1ml),

d. 100mM ascorbic acid solution (1ml),

e. DMSO (78.1µl).

And in the final step, 10mM ferrous ammonium sulphate was added to initiate the reaction and set for 5 min time on magnetic stirrer, then 1ml of reaction mixture was added to any of reagents like chloroform, hexane, and ethyl acetate and mix for 1 min and keep aside for 5 min until the two layers separate, then the aqueous layer was removed for the GC-MS analysis.

STANDARD CONDITIONS OF GC-MS:

The instrumental method which is used in this experiment is a standard one and is continued throughout the study. The analysis was carried out in Shimadzu, a Quadra pole gas chromatograph-mass spectrometer (GC-MS) QP 5050 arranged with a data processing system. The important parameters of the instrument are initial oven temperature of 120⁰C, Helium was used as carrier gas, and the flow rate was maintained at 1ml/min, and also a combination of split and split-less injection are used, and the amount of sample injected was 1µl. The capillary column which was used in GC-MS is of 30m, internal diameter of 0.25mm and film thickness 0.25µm which consists of polydimethylsiloxane (5% phenyl) stationary phase. The initial column temperature was maintained at 100⁰C, after the sample injection the purge activation time was 3 min, and the column was maintained for 2 min, increased at 10⁰C/min to 320⁰C and was hold for 3 min. Both the temperature of the detector and also the ion source are maintained at 250⁰C. The electron ionization mass spectra were obtained over a typical scan range of 50-400m/z and the retention time of the chromatogram was adjusted from 3 min to 15 min.

Spin trapping of ^.CH₃ using 3-Carboxy-Proxyl with GC/MS:

Using the standard Fenton`s reaction conditions in section.1, here 25mM Carboxy-Proxyl (1 ml) was used as the spin trapping agent, and the reaction mixture was mixed for 5min, then 1ml of this was extracted with 1 ml of ethyl acetate and mixed for 1 min and kept aside for 5 min for the separation of the two layers, then the aqueous layer was removed and the layer that consists of all the samples was injected (1 µl) into GC/MS.

Spin trapping of ^.CD₃using 3-Carboxy-Proxyl with GC/MS:

Using the standard Fenton’s reaction conditions in section.1, but instead of using DMSO we used d₆-DMSO, here 25mM 3-Carboxy-Proxyl (1 ml) was used as the spin trapping agent, and the reaction mixture was mixed for 5min, then 1ml of this was extracted with 1 ml of ethyl acetate and kept aside for 5 min for the separation of two layers, then the aqueous layer was removed and the layer that consists of all the samples was injected (1 µl) into GC/MS.

Oxidation of DMSO using Fenton reaction but without H₂O₂:

In this experiment, H₂O₂was not used and the remaining all the samples are same as mentioned before in section.1 for Fenton reaction. Here 25mM 3-Carboxy-Proxyl (1 ml) was used as the spin trapping agent, and all the samples are mixed for 5 min, then 1 ml of reaction mixture was extracted with 1ml of ethyl acetate, for the separation of two layers it was kept aside for 5 min, and the aqueous layer was removed, the layer that contains reaction mixture was injected (1µl) into GC/MS.

Oxidation of DMSO using Fenton reaction but without iron (Fe^II)

From the standard reaction conditions for the Fenton reaction in section.1, iron (Fe^II) was removed and 25mM 3-Carboxy-Proxyl was used as spin trapping, all the samples are mixed for 5 min, then 1 ml of the reaction mixture was extracted with 1 ml of ethyl acetate, then after 1 min shaking it was kept aside 5 min for the separation of two layers, the aqueous layer was removed and the layer that contains all the samples was injected (1µl) into GC/MS.

Spin trapping of ^.CH₃ using 3-Carbamoyl-Proxyl with GC/MS:

Using the standard condition which was mentioned before for Fenton reaction in section.1, here 25mM 3-Carbamoyl-Proxyl (1 ml) was used as spin trapping agent and by adding all the samples, mixed for 5 min and then the reaction mixture was extracted with ethyl acetate (1 ml), and kept aside for 5 min for the separation of two layers. The aqueous layer was removed, and the layer which was containing all the samples was injected (1 µl) into the GC/MS.

Spin trapping of ^.CD₃using 3-Carbamoyl-Proxyl with GC/MS:

Using the standard condition which was mentioned before for Fenton reaction in section.1, but we used d₆-DMSO instead of DMSO, and here 25mM 3-Carbamoyl-Proxyl (1 ml) was used as spin trapping agent, and by adding all the samples, mixed for 5 min and then the reaction mixture was extracted with ethyl acetate (1 ml), and kept aside for 5 min for the separation of two layers. The aqueous layer was removed, and the layer which was containing all the samples was injected (1 µl) into the GC/MS.

Spin trapping of CH₃-CH^.-OH Using 3-Carbamoyl-Proxyl with GC/MS:

In this experiment, ethanol was used instead of the DMSO, and the remaining standard conditions are same as Fenton reaction, and 25mM 3-Carbamoyl-Proxyl (1 ml) was used as spin trapping agent, all the samples are mixed for 5 min, then 1 ml of the reaction mixture was extracted with 1 ml of ethyl acetate, and mixed for 1 min, then for the separation of two layer this was kept aside for 5 min. The aqueous layer was removed and the layer that consists of the all the samples was injected (1 ml) into the GC/MS.

RESULTS AND DISCUSSION:

Spin trapping of ^.CH₃ using 3-Carboxy-Proxyl with GC/MS analysis:

After the injection of extracted reaction mixture, a chromatogram (Fig-1) was obtained with a sharp peak which corresponds to the product peak at t_r = 6.258, with respect to the mass spectra (Fig-2).

Chromatograms:

Fig-1 Chromatogram obtained for the extracted product from GC/MS analysis

Mass Spectra’s:

Fig-2 Full mass spectrum of peak at t_r = 6.258

The compound that was retained at 6.258, was expected to be 3-Carboxy-Proxyl/^.CH₃ adduct [M_r = 201], which was the assumed product, and assigned as I, the ion peak m/z = 186, was assigned as II due to the loss of methyl group from I, the ion peak m/z = 140, was assigned as III, due to the loss of COOH from II. The ion peak m/z = 110 was denoted by IV, due to the loss of two methyl groups from III (Fig-3).

Fragmentations:

Fig-3 Fragmentation of 3-Carboxy-PROXYL/^.CH₃ adduct in GC/MS analysis

Spin trapping of^. CD₃ using 3-Carboxy-Proxyl with GC/MS:

Injection of the extracted reaction mixture a chromatogram (Fig-4) was obtained with a strong peak which corresponds to the product peak retention time at t_r = 6.292 with respect to the mass spectra (Fig-5).

Fig-4 Chromatogram obtained for the extracted product from GC/MS analysis

Fig-5 Full mass spectrum of peak at t_r = 6.292

The compound that was retained at 6.292 was expected to be 3-Carboxy-Proxyl/CD₃ adduct [M_r = 204], which was the expected product and was assigned as I, the ion peak m/z = 189, was denoted by II, due to the loss of one methyl group from I, The ion peak m/z = 143, was assigned as III, due to the loss of COOH from II, the ion peak obtained at m/z = 110, was assigned as IV, due to the loss of one methyl and CD₃ radical from III(fig-6).

Fig-13 Chromatogram obtained for the extracted product from GC/MS analysis

Fig-14 Full mass spectrum of peak at t_r = 9.458

Oxidation of DMSO using Fenton reaction but without H₂O₂:

After the injection of extracted reaction mixture which doesn’t consist of hydrogen peroxide (H₂O₂), a chromatogram was obtained with no peak corresponding to the final product. This is because peroxide plays an important role in the formation of hydroxyl radicals but without that there will be no interaction between DMSO and hence no trapping of methyl radicals are observed.

Oxidation of DMSO using Fenton reaction but without iron (Fe^II):

After the injection of extracted reaction mixture, a chromatogram was obtained with no peak corresponding to the final product this is due to the absence of iron. In the absence of Fe^II , the hydrogen peroxide will not oxidize and hence no production of hydroxyl radicals so finally we cannot see the final product peak.

CONCLUSION:

GC/MS analysis was used in this study, to detect a novel product of the reaction between the ^.CH₃ and the spin trapping agent’s 3-Carboxy-Proxyl and 3-Carbamoyl–Proxyl and thus provide an accurate means of measuring hydroxyl radicals. The alternative method GC/MS plays an important role in the identification and measurement of free radicals. By using the Fenton`s reagent the hydroxyl radicals was produced that is iron-EDTA reacts with H₂O₂ in the presence of ascorbic acid. In this study we have used a well known reaction, in which hydroxyl radical [^.OH] reacts with dimethyl sulfoxide [DMSO] where the OH radical was converted to methyl radical [^.CH₃]. The spin trapping agents 3-Carboxy-Proxyl and 3-Carbamoyl–Proxyl used for the trapping of methyl radicals and the other free radical adducts could be detected by GC/MS analysis. The results that are obtained shows that both the spin trapping agent is important for the reaction to complete and proves the DMSO reacts with hydroxyl radical to produce methyl radicals which can be trapped by the spin trapping agents 3-Carboxy-Proxyl and 3-Carbamoyl–Proxyl. In this experiment, the results obtained by using deuteriated DMSO shows a strong evidence to demonstrate 1, 3-addition product.

The results obtained by the GC/MS analysis shows that 3-Carboxy-Proxyl and 3-Carbamoyl–Proxyl have a potential to work as spin trapping agents by trapping methyl radical, and also with deuteriated DMSO it traps CD₃ radicals. This shows an ample evidence to work as spin traps.

The overall aim of the work was to investigate the biomarkers which are oxygen free radicals produced using GC/MS. By indirectly trapping and searching methyl radicals and analyzing using GC/MS, it has been shown that the hydroxyl radicals are monitored. By using this technique, we detected hydroxyl radical indirectly by trapping methyl radicals.

ACKNOWLEDGEMENT:

We wish to thank Natco Pharmaceuticals, Hyderabad for the gift sample and, Vimtha Laboratories, Hyderabad for providing the GC/MS to carrying out spectral analysis. Finally we are grateful to the management of SRK foundation for providing necessary facilities.

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