Rapid and fast strategy for the determination of glutathione in the presence of vitamin B₆ in biological and pharmaceutical samples using a nanostructure based electrochemical sensor

Abstract

Cyclic voltammetry (CV), square wave voltammetry (SWV), electrochemical impedance spectroscopy (EIS) and double potential step chronoamperometry were used to investigate the electrochemical oxidation of glutathione (GSH) at a chemically modified electrode prepared by incorporating acetylferrocene (AF) and NiO nanoparticles (NiO/NPs) into a carbon paste matrix. For the mixture containing GSH and vitamin B₆, the peak potentials were well separated from each other. Under optimal conditions in cyclic voltammetry, linear ranges spanned a GSH concentration from 0.2 μM to 350.0 μM and the detection limit was 0.09 μM at a signal-to-noise ratio of 3. The novel sensor was successfully used for the voltammetric determination of the GSH in real samples with satisfactory results.

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1. Introduction

Glutathione (GSH) is a simple biological molecule that is constantly produced naturally in the human body. GSH protects DNA from oxidative stress during cell division, which allows for DNA synthesis. When DNA is mutated by a free radical that steals an electron from DNA, GSH repairs the mutated DNA by providing an electron to the DNA.¹ Normally GSH is recycled in the body, especially when the toxic load becomes very high. Low levels of GSH are commonly observed in wasting and negative nitrogen balance,² as seen in cancer, trauma, burns, HIV/AIDS, sepsis, and athletic overtraining.³ Therefore, the determination of GSH is very important in the human body. Many analytical methods have been reported for its determination in the human body, which include titrimetry,⁴ spectrophotometry,⁵ spectrofluorimetry,⁶ HPLC,⁷ capillary zone electrophoresis,⁸ flow injection analysis,⁹ and electrochemical methods.^10–15

Vitamin B₆ with other medications such as doxylamine or metoclopramide has been used to treat nausea and vomiting in early pregnancy for decades.¹⁶ Vitamin B₆ is important in the conversion of tryptophan to serotonin in the human body. Takeuchi et al. showed that the glutathione levels and related enzyme activities can be effective in vitamin B₆-deficiency.¹⁷ Therefore, the investigation of GSH and vitamin B₆ levels is very important in biological samples such as those from the human body.

Among the many different analytical methods, electrochemical techniques have good ability for the simultaneous determination of biological and environmental compounds.^18–23 Electrochemical methods based on modified electrodes have attracted significant attention due to their convenience, easy operation, good sensitivity, high selectivity and reproducibility.^22–30

To the best of our knowledge, a study for the simultaneous determination of GSH and vitamin B₆, especially using modified electrodes, has not been reported to date. The main object of this study was to simultaneously detect GSH and vitamin B₆ using a novel modified electrode in biological and pharmaceutical samples. In the present study, cyclic voltammetry, chronoamperometry, square wave voltammetry and electrochemical impedance spectroscopic techniques were used to study the electrochemical behavior of GSH and vitamin B₆ on a novel AF/NiO/NPs/CPE electrode. The detection limit, linear dynamic range, and sensitivity to GSH with the AF/NiO/NPs/CPE are comparable to, and even better than, those recently developed using voltammetric methods.

2. Experimental

2.1. Materials and instrumentation

Glutathione, vitamin B₆, sodium phosphate monobasic, sodium hydroxide, sodium phosphate dibasic anhydrous, sodium phosphate, and phosphoric acid were purchased from Sigma-Aldrich and were used as received without any further purification. Graphite powder, nickel nitrate and paraffin oil were purchased from Merck. Double distilled water was used for the preparation of all the solutions. Phosphate buffer solutions (0.1 mol L⁻¹) (PBS) with different pH values were used for pH optimization study. NiO/NPs were prepared by a reported procedure.³¹

X-ray powder diffraction (STOE diffractometer with Cu–Kα radiation) was performed to study the synthesis and determine the size of the NiO/NPs. A voltammetric investigation was performed with an electroanalytical system, Autolab PGSTAT 12, potentiostat/galvanostat, connected to a three-electrode cell, Metrohm Model 663 VA stand, linked with a computer (Pentium IV) and with Autolab software. The system was run on a PC using GPES and FRA 4.9 software.

2.2. Preparation of real samples

For real sample analysis in biological conditions, human urine and erythrocyte samples were taken voluntarily from a healthy woman immediately before the experiments and informed consent was obtained from her. All the experiments were performed in compliance with the relevant laws and institutional guidelines, and also institutional committees approved the experiments.

The erythrocyte pellets were hemolysed with water (1 : 1, v/v). For protein precipitation, the hemolysate was mixed with 5-sulfosalicylic acid (10%, m/v) in the ratio of 2 : 1 (v/v). This mixture was centrifuged for 10 min at 4000 rpm.

The tablet (Chongqing Yaoyou Pharmaceutical Co., Ltd) solution was prepared by completely grinding and homogenizing ten tablets of GSH, labeled 100 mg per tablet. The required amount of the powder was accurately weighed and dissolved in 100 mL PBS with ultrasonication.

The human urine samples were analyzed after 3.0 h of their sampling, except when stated otherwise. In the first step, the urine sample was centrifuged (1500 rpm, room temperature) and then diluted two-times with PBS without any further pretreatment.

2.3. Sensor preparation

1.0% (w/w) mediator spiked graphite and NiO/NPs was made by dissolving the given quantity of AF in diethyl ether and mixing it with 84.0% (w/w) of graphite powder and 15.0% (w/w) NiO/NPs with a mortar and pestle. Diethyl ether was evaporated by stirring and a mixture of NiO/NPs, NiO/NPs spiked carbon powder plus paraffin oil was blended by mixing with hand. The obtained paste was inserted at the bottom of a glass tube. Finally, an electrical connection was implemented with a copper wire lead fitted into the glass tube.

3. Results and discussion

A typical X-ray pattern collected for the synthesised nanoparticle is presented in Fig. S1 (ESI†). Peaks appear at 2θ = 37.63°, 43.80°, 63.09°, 75.72° and 79.42°, which correspond to the diffraction patterns of (1 1 1), (2 0 0), (2 2 0), (3 1 1) and (2 2 2), respectively. Some other peaks are relative to NaNO₃ adsorbed on the surface of the nanoparticles. The diameter of the synthesized NiO/NPs was calculated using the Debye–Scherrer equation. The average particle size of the synthesised nanoparticle was found to be 30 nm, which was derived from the FWHM of a more intense peak corresponding to the (2 0 0) plane located at 43.80° using Scherrer's formula.

Although the ferrocene/ferrocenium redox couple is independent of pH, it is known that the electrochemical behavior of GSH depends on the pH value. For pH optimization, we studied the electrooxidation behavior of GSH in 0.1 M PBS with various pH values (4.0 < pH < 7.0) at the surface of the modified electrode. Results show that the maximum electrocatalytic current was obtained at pH = 6.0. All the subsequent electrochemical experiments were performed at this optimized pH value. Moreover, a pair of quasi-reversible peaks were observed at E_pa = 0.66 V and E_pc = 0.54 V vs. Ag/AgCl (scan rate 0.15 V s⁻¹). The half-wave potential (E_1/2) was 0.06 V vs. Ag/AgCl and ΔE_p(E_pa − E_pc) was 0.14 V (not shown). The electrode process was quasi-reversible with ΔE_p greater than the expected value (59/n mV) for a reversible system. At low scan rates, the ferrocenium formed left the diffusion layer and the reverse signal could not be seen under these conditions.

Fig. 1 depicts the electrochemical oxidation responses of 600.0 μM GSH at the AF/NiO/NPs/CPE (curve c), AF/CPE (curve b), NiO/NPs/CPE (curve d), and CPE (curve e). As shown, the anodic peak potential for GSH oxidation at the AF/NiO/NPs/CPE (curve c) and AF/CPE (curve b) was about 610 mV, while at the NiO/NPs/CPE (curve d), the peak potential was about 860 mV. At the CPE, the oxidation peak potential was about 890 mV of GSH (curve b). By comparing the present data, it was concluded that the best electrocatalytic oxidation effect for GSH was observed at the AF/NiO/NPs/CPE (curve c). For example, cyclic voltammetry results showed that the oxidation peak potential of GSH oxidation at the AF/NiO/NPs/CPE (curve c) shifted by about 250 mV and 280 mV toward negative values when compared with that at the NiO/NPs/CPE (curve d) and the bare carbon paste electrode (curve e), respectively. Under the same conditions, when comparing the oxidation signal of GSH at the AF/NiO/NPs/CPE (curve c) and AF/CPE (curve b), a dramatic enhancement of the oxidation peak current at the AF/NiO/NPs/CPE relative to that obtained at the AF/CPE was observed. The present data clearly shows that the combination of NiO/NPs and AF definitely improves the characteristics of GSH oxidation. The suitable electronic properties of NiO/NPs provided the ability to promote charge transfer reactions when used as an additive to an electrode surface.

Fig. 1 Cyclic voltammograms of (a) the buffer solution at AF/NiO/NPs/CPE, (b) 600 μM GSH at AF/CPE, (c) 600 μM GSH at AF/NiO/NPs/CPE, (d) 600 μM GSH at NiO/NPs/CPE, and (e) 600 μM GSH at CPE. Conditions 0.1 mol L⁻¹ PBS (pH 6.0), scan rate of 20 mV s⁻¹.

The dependence of the oxidation current response on the potential scan rate was evaluated by varying the scan rate during the electrocatalytic oxidation of GSH at the surface of the modified electrode. Fig. 2 (inset) depicts the cyclic voltammograms observed for the oxidation of 300 μM GSH at AF/NiO/NPs/CPE at different scan rates from 1 to 17 mV s⁻¹. The results presented in Fig. 2 confirmed that there is a linear relationship between the oxidation peak current and the square root of the scan rate. This linear relationship indicates that the oxidation of GSH at AF/NiO/NPs/CPE is a diffusion-controlled process.^32–34

Fig. 2 Plot of I_pa versus v^1/2 for the electro-oxidation of 300.0 μM GSH at various scan rates of (a) 1.0, (b) 4.0, (c) 6.0, (d) 8.0, (e) 11.0, (f) 15.0 and (g) 17.0 mV s⁻¹ in 0.1 mol L⁻¹ PBS (pH 6.0) at AF/NiO/NPs/CPE. Inset; cyclic voltammograms of 300.0 μM GSH at various scan rates.

The Tafel plot was used to obtain information about the rate-determining step (Fig. 3) in the electro-oxidation of GSH at the surface of AF/NiO/NPs/CPE. The slopes of the obtained plots were equal to 2.3RT/n(1 − α)F, which were observed to be 0.0987 and 0.0997 V per decade for scan rates 4.0 and 11 mV s⁻¹, respectively. Using the abovementioned data, we obtained the mean value of α equal to 0.4.

Fig. 3 Tafel plot for AF/NiO/NPs/CPE in 0.1 mol L⁻¹ PBS (pH 6.0) with a scan rates of 4.0 and 11.0 mV s⁻¹ in the presence of 300.0 μM GSH.

The chronoamperometric measurements of GSH at AF/NiO/NPs/CPE were carried out by setting the working electrode potential at 0 and 0.75 V as the first and second potential versus Ag/AgCl/KCl_sat for the various concentrations of GSH in PBS (Fig. 4A), respectively. For an electroactive material (GSH in this case) with a diffusion coefficient of D, the oxidation current observed for an electrochemical reaction at the mass transport limited condition is described by the Cottrell equation.³⁵ Experimental plots of I vs. t^−1/2 were acquired with the best fits for different concentrations of GSH. The slopes of the resulting straight lines were then plotted vs. GSH concentration (Fig. 4B). Using the resulting slope and Cottrell equation the mean value of the D was found to be 9.31 × 10⁻⁵ cm² s⁻¹.

Fig. 4 (A) Chronoamperometry curves obtained at AF/NiO/NPs/CPE in the absence (a) and in the presence of (b) 450 and (c) 550 μM GSH in a buffer solution (pH 6.0). (B) Cottrell plots obtained from the chronoamperometry curves. Inset (C) Dependence of I_C/I_L on the t^1/2 derived from data in curves (A).

The rate constant for the chemical reaction between GSH and redox sites in AF/NiO/NPs/CPE, k_h, can be evaluated by chronoamperometry, according to the method of Galus:³⁶

I_C/I_L = γ^1/2π^1/2 = π^1/2(k_hC_bt)^1/2 (1)

In the abovementioned equation, I_C is the electro-catalytic current of GSH at the modified electrode, I_L is the current in the absence of GSH, and t is the time elapsed (s). Based on the slope of the Fig. 4C, k_h can be obtained for a given GSH concentration. Using the values of the slopes, the average value for k_h was found to be 6.04 × 10² mol⁻¹ L s⁻¹.

For further investigation, we used EIS for the study of the electrocatalytic interaction of GSH at the surface of AF/NiO/NPs/CPE (Fig. 5). For this aim, we compare the value of the charge transfer coefficient in the absence and presence of 400 μM GSH at the surface of the modified electrode. In the presence of GSH, the diameter of the semicircle decreases, confirming the electrocatalytic capability of the mentioned electrocatalyst for the oxidation of GSH. This is due to the instant chemical reaction of GSH with the high-valence AF species. The equivalent electrical circuit (from this system) that is compatible with the impedance spectra is shown in the inset of Fig. 5. R_s (the solution/electrolyte resistance), R_ct (charge-transfer resistance), Z_w, (Warburg impedance) related to the semi-infinite linear diffusion, and C_dl (double layer capacitance) are the elements of this circuit. The appearance of both kinetic (R_ct) and diffusion (Z_w) domains indicates mixed electrode reactions.

Fig. 5 Nyquist diagrams of AF/NiO/NPs/CPE (a) in the absence and (b) in the presence of 400 μM GSH. Inset: equivalent circuit for the system.

Square wave voltammetry (SWV) was used to determine GSH under the optimum conditions. Responses were linear with GSH concentrations ranging from 0.2 μM to 350 μM and a current sensitivity of 0.2011 μA μM⁻¹. The detection limit (3σ) was 0.09 μM.

The simultaneous determination of GSH and vitamin B₆ is one of the most important applications of the proposed sensor. This study involved the simultaneous determination of GSH and vitamin B₆ by simultaneously changing the concentrations of GSH and vitamin B₆ and recording the SWVs. Results showed two well-defined oxidation peaks with a separation of 309 mV (Fig. 6 inset). Current sensitivities towards GSH in the absence and presence of the other compound were found to be 0.2011 μA μM⁻¹ (in the absence of vitamin B₆) and 0.2035 μA μM⁻¹ (in the presence of vitamin B₆) (Fig. 6). This point is very interesting to note that the sensitivities of the AF/NiO/NPs/CPE towards GSH in the absence and presence of vitamin B₆ were virtually the same, which indicates that the oxidation processes of GSH and vitamin B₆ at the AF/NiO/NPs/CPE are independent and that simultaneous or independent measurements of the two compounds are, therefore, possible without any interference. The SWV is a highly sensitive method compared to cyclic voltammetric method. It is common for SWV peaks to have peak potentials that occur at less positive values than those of CV.

Fig. 6 The oxidation plots of the electrocatalytic peak current as a function of GSH concentration. Inset: SWVs of AF/NiO/NPs/CPE in 0.1 M PBS (pH 6.0) containing different concentrations of GSH–vitamin B₆ in μM. (a–e): 45.0 + 80.0; 90.0 + 120.0; 120.0 + 150.0; 160.0 + 200.0 and 190.0 + 220.0, respectively.

Long-term stability for any proposed sensor is one of the most important properties. The stability of AF/NiO/NPs/CPE was investigated by square wave voltammetry in the presence of GSH. The oxidation response currents can retain almost constant values upon continuous eight cyclic sweeps over the applied potential ranging from 0.20 to +0.70 V. After storage in the refrigerator at 4 °C for 30 days, the potentials of oxidation peak in square wave voltammetry remained at the same positions and the peak currents decreased by only about 3.1% to that of the initial values before storage. The relative standard deviation (RSD%) for nine successive assays was 1.9%. When using four different electrodes, the RSD for five measurements was 2.7%.

In continuing to evaluate the selectivity of AF/NiO/NPs/CPE in the determination of GSH, the influence of different foreign species on the determination of 15.0 μM GSH was investigated. The tolerance limit was taken as the maximum concentration of foreign substances that caused an approximate relative error of ±5%. The obtained results are shown in Table 1. The results in Table 1 demonstrate that AF/NiO/NPs/CPE has good selectivity for GSH analysis.

Table 1 Interference study for the determination of 15.0 μM GSH under the optimized conditions (pH 6.0)

Species	Tolerance limits (Wspecies/WGSH)
a After addition of 1 mmol L^−1 ascorbate oxidase.
Alanine, phenyl alanine, tryptophan, methanol, glycine, L-threonine, and L-isoleucine, Ca^2+, Br^−, Mn^2+, K^+, Li^+, sucrose, lactose and fructose	1000
Urea, ascorbic acid^a, thiourea	900
Starch	Saturation

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Finally, we checked the ability of the AF/NiO/NPs/CPE as a new sensor for the determination of GSH in pharmaceutical and biological samples. The standard addition method was used for this investigation. The obtained data was compared with other published methods as well. The results are given in Table 2, which confirm that the AF/NiO/NPs/CPE retained its efficiency for the determination of GSH in real samples.

Table 2 Determination of GSH in different types of real samples^a

Sample	Added (μM)	Proposed method (μM)	Elman method^37 (μM)	Fex	Ftab,(0.05);2,2	tex	ttab (98%)
a Fex calculated F value; reported F value from F-test table with 95% confidence level and 2/2 degrees of freedom tex calculated t; ttab (95%); reported t value from Student's t-test table with 95% confidence level.
Hemolysed erythrocyte	—	5.87 ± 0.33	6.01 ± 0.59	8.5	19	2.6	3.8
	—	8.52 ± 0.52	8.95 ± 0.82	—	—	—	—
Urine		<LOD	<LOD	—	—	—	—
	50.0	51.05 ± 0.75	50.85 ± 0.89	10.5	19	3.1	3.8
	100.0	102.15 ± 2.45	102.37 ± 2.65	—	—	—	—
Tablet	10.0	9.77 ± 0.50	10.56 ± 0.68	7.3	19	2.8	3.8
	20.0	20.55 ± 0.74	20.76 ± 0.85	—	—	—	—

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4. Conclusion

The AF/NiO/NPs/CPE was prepared and used for the study of the electrochemical oxidation of GSHs. The AF/NiO/NPs/CPE showed excellent electrocatalytic activity for the electro-oxidation of GSH. The SWV currents of GSH at AF/NiO/NPs/CPE increased linearly with the GSH concentration in the range from 0.2 to 350.0 μM with a detection limit of 0.09 μM. Potential difference between GSH and vitamin B₆ was determined to be of 309 mV, which was large enough to simultaneously determine GSH and vitamin B₆. Finally, AF/NiO/NPs/CPE was used for the determination of GSH in some real samples.

Acknowledgements

The authors are grateful to University of Kashan for supporting this work by Grant no. (434066-5).

References

1. http://www.immunehealthscience.com/glutathione.html, 24, April, 2014.
2. D. Wulf and H. Eggert, FASEB J., 1997, 11, 1077–1089.
3. http://en.wikipedia.org/wiki/Glutathione, 24, April, 2014.
4. P. Nagendra, H. S. Yathirajan and K. S. Rangappa, J. Soc. Chem. Ind., 2002, 79, 602–604.
5. M. Raggi, L. Nobile and A. G. Giovannini, J. Pharm. Biomed. Anal., 1991, 9, 1037–1040.
6. S. C. Liang, H. Wang, Z. M. Zhang, X. Zhang and H. S. Zhang, Anal. Chim. Acta, 2002, 451, 211–219.
7. A. E. Katrusiak, P. G. Paterson and H. Kamencic, J. Chromatogr. B, 2001, 758, 207–212.
8. E. Causse, P. Malatray, R. Calaf, P. Chariots, M. Candito, C. Bayle, P. Valdiguie, C. Salvayre and F. Couderc, Electrophoresis, 2000, 21, 2074–2079.
9. A. A. Ensafi, T. Khayamian and F. Hasanpour, J. Pharm. Biomed. Anal., 2008, 48, 140–144.
10. J. B. Raoof, R. Ojani and H. Karimi-Maleh, J. Appl. Electrochem., 2009, 39, 1169–1175.
11. A. A. Ensafi, M. Taei, T. Khayamian, H. Karimi-Maleh and F. Hasanpour, J. Solid State Electrochem., 2010, 14, 1415–1423.
12. J. B. Raoof, R. Ojani, H. Karimi-Maleh, M. R. Hajmohamadi and P. Biparva, Anal. Methods, 2011, 3, 2637–2643.
13. H. Karimi-Maleh, F. Tahernejad-Javazmi, A. A. Ensafi, R. Moradi, S. Mallakpour and H. Beitollahi, Biosens. Bioelectron., 2014, 60, 1–7.
14. H. Karimi-Maleh, F. Tahernejad-Javazmi, V. K. Gupta, H. Ahmar and M. H. Asadi, J. Mol. Liq., 2014, 196, 258–263.
15. R. Moradi, S. A. Sebt, H. Karimi-Maleh, R. Sadeghi, F. Karimi, A. Bahari and H. Arabi, Phys. Chem. Chem. Phys., 2013, 15, 5888–5897.
16. P. Sheehan, Aust Fam Physician, 2007, 36, 698–701.
17. F. Takeuchi, S. Izuta, R. Tsubouchi and Y. Shibata, J. Nutr., 1991, 121, 1366–1373.
18. B. J. Sanghavi, P. K. Kalambate, S. P. Karna and A. K. Srivastava, Talanta, 2014, 120, 1–9.
19. B. J. Sanghavi and A. K. Srivastava, Electrochim. Acta, 2011, 56, 4188–4196.
20. B. J. Sanghavi, S. Sitaula, M. H. Griep, S. P. Karna, M. F. Ali and N. S. Swami, Anal. Chem., 2013, 85, 8158–8165.
21. B. J. Sanghavi and A. K. Srivastava, Electrochim. Acta, 2010, 55, 8638–8648.
22. L. M. Yola, N. Atar, Z. Üstündağ and A. O. Solak, J. Electroanal. Chem., 2013, 698, 9–16.
23. M. L. Yola, N. Atar, M. S. Qureshi, Z. Üstündağ and A. O. Solak, Sens. Actuators, B, 2012, 171–172, 1207–1215.
24. M. L. Yola, V. K. Gupta, T. Eren, A. E. Sen and N. Atar, Electrochim. Acta, 2014, 120, 204–211.
25. M. L. Yola and N. Atar, Electrochim. Acta, 2014, 119, 24–31.
26. H. Karimi-Maleh, P. Biparva and M. Hatami, Biosens. Bioelectron., 2013, 48, 270–275.
27. S. Gheibi, H. Karimi-Maleh, M. A. Khalilzadeh and H. Bagheri, J. Food Sci. Technol., 2015, 52, 276–284.
28. H. Karimi-Maleh, F. Tahernejad-Javazmi, N. Atar, M. L. Yola, V. K. Gupta and A. A. Ensafi, Ind. Eng. Chem. Res., 2015, 54, 3634–3639.
29. N. Atar, T. Eren, M. L. Yola, H. Karimi-Maleh and B. Demirdögena, RSC Adv., 2015, 5, 26402–26409.
30. M. Elyasi, M. A. Khalilzadeh and H. Karimi-Maleh, Food Chem., 2013, 141, 4311–4317.
31. H. Karimi-Maleh, M. Salimi-Amiri, F. Karimi, M. A. Khalilzadeh and M. Baghayeri, J. Chem., 2013, 2013, 946230.
32. M. Najafi, M. A. Khalilzadeh and H. Karimi-Maleh, Food Chem., 2014, 158, 125–131.
33. H. Karimi-Maleh, S. Rostami, V. K. Gupta and M. Fouladgar, J. Mol. Liq., 2015, 201, 102–107.
34. V. K. Gupta, H. Karimi-Maleh and R. Sadegh, Int. J. Electrochem. Sci., 2015, 10, 303–316.
35. A. J. Bard and L. R. Faulkner, Electrochemical Methods Fundamentals and Applications, 2nd edn, Wiley, New York 2001.
36. Z. Galus, Fundumentals of Electrochemical Analysis, Ellis Horwood, New York, 1976.
37. G. L. Ellman, Arch. Biochem. Biophys., 1959, 82, 70–77.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra08433d

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