A Simple HPLC-UV Method for the Determination of Glutathione in PC-12 Cells.

A highly sensitive and simple HPLC-UV method was developed and validated for the assay of glutathione (GSH) in PC-12 cells. Glutathione is a major intracellular antioxidant having multiple biological effects, best known for its cytoprotective effects against cell damage from reactive oxygen species and toxic reactive metabolites and regulating the cellular redox homeostasis. Due to its own sulfhydryl (SH) group, GSH readily reacts with Ellman's reagent to form a stable dimer which allows for quantitative estimation of GSH in biological systems by UV detection. The separation was achieved using a C8 column with a mobile phase consisting of phosphate buffer adjusted to pH 2.5 (mobile phase A) and acetonitrile (mobile phase B), running in a segmented gradient manner at a flow rate of 0.8 mL/min, and UV detection was performed at 280 nm. The developed HPLC-UV method was validated with respect to precision, accuracy, robustness, and linearity within a range of 1-20 μg/mL. Limit of detection (LOD) and limit of quantification (LOQ) were 0.05 and 0.1 μg/mL, respectively. Furthermore, the method shows the applicability for monitoring the oxidative stress in PC-12 cells.

1. Introduction

Glutathione (GSH) is chemically known as (2S)-2-amino-4-[[(1R)-[(carboxymethyl) carbamoyl]-2-sulfanylethyl] carbamoyl] butanoic acid. GSH is a tripeptide (Figure 1), often considered as the mother of all antioxidants and is present in almost every cell. Because GSH exists within the cells, it is in a prime position to neutralize free radicals. The strong antioxidant effect of GSH helps keep cells running smoothly and also helps the liver to remove chemicals that are foreign to the body such as drugs/pollutants [1, 2]. In addition, GSH has the potential to fight almost any disease; particularly those associated with ageing, since free radical damage is the cause of many of the diseases of old age. GSH is nucleophilic at the sulfur and attacks poisonous electrophilic conjugate acceptors. Thiol groups are kept in a reduced state at a concentration of approximately ~5 mM in animal cells. In effect, GSH reduces any disulfide bond formed within cytoplasmic proteins to cysteines by acting as an electron donor. In the process, GSH is converted to its oxidized form glutathione disulfide (GSSG). Glutathione is found almost exclusively in its reduced form, since the enzyme that reverts it from its oxidized form, GSSG, is constitutively active and inducible upon oxidative stress. In fact, the ratio of GSH to GSSG within cells is often used scientifically as a measure of cellular toxicity [3-5].

Figure 1

Reaction of Ellman's reagent with glutathione.

In healthy cells and tissue, more than 90% of the total glutathione pool is in the reduced form and less than 10% exists in the disulfide form [6]. An increased GSSG-to-GSH ratio is considered indicative of oxidative stress. Several methods were reported earlier to estimate the amount of GSH present in biological samples and commercial products using HPLC [7, 8], capillary zone electrophoresis [9-11]. However, a simple HPLC-UV method for quantification of GSH in PC-12 cells and its role in cellular stress is yet to be found. This study attempts to develop and validate a simple HPLC-UV method for the determination of GSH in PC-12 cells.

2. Materials and Method

2.1. Chemicals and Reagents

Glutathione (GSH-reduced form) was purchased from Acros (USA), 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB or Ellman's reagent), Dulbecco's Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), acetonitrile (ACN), and potassium monobasic phosphate were obtained from Fisher Scientific Co., and Whatman® Grade 1 Qualitative Filtration Paper, phosphoric acid, and tri-chloroacetic acid (TCA) were obtained from Sigma-Aldrich (USA). In-house purified deionized (DI) water was used throughout the investigation.

2.2. HPLC Instrument and Separation Parameters

Hitachi LaChrom series LC system, consisting of an L-7100 pump, an L-7200 autosampler, an L-7400 UV detector set at a wavelength of 280 nm, and D-7000 interface with system manager data acquisition software (version 5.0), was used throughout the study. The chromatographic separation was achieved using an Agilent Eclipse XDB C8 (150 × 4.6 mm, 5 μ) column, the optimized method used a segmented gradient mobile phase with phosphate buffer at pH = 2.5 as solvent (A) and ACN as solvent (B), and the gradient program is shown in Table 1. The sample volume for injection was 50 μL and the total run time was 20 min.

Table 1

Summary of gradient program.

Time (min)	% mobile phase (A) Phosphate buffer	% mobile phase (B) ACN	Flow rate (mL/min)
0	90	10	0.8
4.5	60	40	0.8
4.6	60	40	0.5
14.0	60	40	0.5
14.1	60	40	0.8
20.0	90	90	0.8

2.3. Preparation of Standard Solution

A stock solution of GSH at 100 μg/mL concentrations was prepared by weighing 10 mg of GSH in 100 mL volumetric flask and making up the volume with DI water. The stock solution was stored at 4°C and appropriate dilutions of GSH were prepared to make working standards of 0.1, 0.5, 1, 2, 5, 10, and 20 μg/mL of various validation studies. 500 μg/mL of Ellman's reagent was prepared by accurately weighing 50 mg of reagent in 100 mL of methanol and stored at 4°C.

2.4. Sample Preparation and Withdrawal of GSH from PC-12 Cells

Cells (1 × 106 cells/sample) were cultured in DMEM supplemented with 10% FBS at 37°C for 24 h, followed by treatment with either MnCl2 (5 mM), CoCl2 (5 mM), methylglyoxal (0.4 mM), or hydrogen peroxide (0.1%) for additional 24 h. Cells grown in DMEM alone were used as controls. Cells were centrifuged at 3,000 rpm for 90 seconds and the supernatant was discarded. The cell pellet was suspended in 10% ice-cold TCA and centrifuged for 15 min at 9,000 ×g. The supernatant was collected and GSH was measured by using HPLC-UV.

2.5. Derivatization of GSH

Since its introduction in 1959, Ellman's reagent has been the favorite reagent for spectrophotometric measurement of protein sulfhydryls. For GSH analysis, an aliquot of 0.5 mL of GSH solution was added to 0.5 mL of 0.5 mM Ellman's reagent solution. The solution was allowed to react for 30 min at 60°C followed by injecting into the HPLC at a flow rate of 0.8 mL/min, the separation was performed at ambient temperature, and the detection was carried at 280 nm. For biological samples, the sample from PC-12 cells was first filtered through Millipore membrane followed by the addition of 0.5 mL Ellman's reagent (0.5 mM).

3. Results and Discussion

3.1. Method Development

From the structure of GSH, it is very clear that the compound is highly polar; however, the compound was found to be insoluble in ACN and methanol and completely soluble in water. In the development of a RP-HPLC method for GSH, it was determined that GSH is not retained in the RP columns; therefore, we choose to derivatize the sample with Ellman's reagent popularly used to quantify thiols [12]. The proposed derivatized compounds (Figure 1) are 2-nitro-5-mercapto-benzoic (NMB) acid and glutathione dimer (GSH Dimer). These compounds have demonstrated some increase in hydrophobicity and were effectively retained in the RP columns, allowing the separation of compounds in the sample to occur. Evidence has shown that the maximum UV-Vis spectrum absorbance for the dimer is at 412 nm. For this experiment, an appropriate wavelength was selected at 280 nm to reduce the baseline disturbances and improve the signal strength. Next step was to optimize the separation conditions, different RP columns were tested, and finally Agilent Eclipse XDB C8 column was selected. The mobile phase consisted of phosphate buffer at pH = 2.5 as solvent (A) and ACN as solvent (B); the main reason to select a pH at 2.5 was to protonate all the free silanols in the column and to reduce their chromatographic activity [13].

A segmented gradient program (Table 1) was used to achieve separation with the retention times (RT) of NMB, GSH Dimer, and DTNB at 7.65, 11.23, and 14.83, respectively (Figure 2). When the developed method was tested on samples extracted from PC-12 cells, no endogenous interfering peaks were observed in the individual blank sample at the RT of GSH biosample, making the developed method of high ruggedness.

Figure 2

Representative chromatogram from 6 replicates is shown. 2-Nitro-5-mercapto-benzoic (NMB) acid, glutathione dimer (GSH Dimer), and Ellman's reagent (DTNB) in GSH raw material.

3.2. Validation of Developed Method

Method validation was performed in terms of system suitability, linearity, precision, accuracy, robustness, and finally sensitivity [14-16].

3.2.1. System Suitability

System suitability tests (SST) are an integral part of liquid chromatography methods. They are used to verify that the resolution and reproducibility of the chromatography system are adequate for the analysis to be done. The system suitability test is a US Food and Drug Administration (FDA) validation requirement [14-16] and is usually considered as a prevalidation requirement (equipment performance qualification test). SST was evaluated by injecting 3 blank samples (diluting solvent), followed by 6 injections of GSH (100 μg/mL). Parameters such as USP plate count were found to be 5878, tailing factor is 1.1, resolution is 2.42 for GSH in PC-12 cells, and repeatability [%Relative Standard Deviation (RSD) of RT and peak areas] was examined and compared against the specifications set for the method. The % RSD was found to be less than 1.0.

3.2.2. Linearity and Sensitivity

Calibration curves were obtained (using least squares method) by plotting the concentration ratio versus the peak area ratio for the analyte (Figure 4). The method showed linearity within the range of 1 to 20 μg/mL with a correlation cost ability greater than 0.998. The LOD was defined as the compound concentration that produces a signal-to-noise (S/N) ratio greater than three and it was found to be 0.05 μg/mL. The limit of quantitation for the assay was evaluated as the concentration ten times to S/N ratio and was found to be 0.1 μg/mL.

Figure 4

Linearity plot of 2-nitro-5-mercapto-benzoic (NMB) acid and glutathione dimer (GSH Dimer).

3.2.3. Precision and Recovery

Injection precision (repeatability) was determined by six injections of standard and also by calculating the system suitability factors. The method precision was carried out by freshly prepared standards and % RSD value was calculated for peak areas. For examining interday precision (reproducibility), the samples were analyzed by second chemist on a different day using a different instrument with help of freshly prepared samples. Satisfactory repeatability and precision were achieved with RSD values within the limits. The acceptance criterion for repeatability (intraday precision) and intermediate (interday precision) RSD should be better than 20% at lower concentrations and better than 15% at higher concentrations (Table 2).

Table 2

Intraday and interday precision studies for the determination of GSH using HPLC-UV.

Added (µg/mL)	IntradayFound (µg/mL) ± SD	RSD(n = 6)	Recovery (%)
20	19.7	1.3	98
40	40.1	2.2	100.25
60	58.8	1.9	98
Interday (n = 6)
20	19.2	1.8	96
40	39.2	1.5	98
60	57.2	2.7	95.3

The recovery of GSH from PC-12 cells was estimated by spiking 20, 40, and 60 μg/mL concentration in six replicates. Six replicate samples containing the same strength of GSH in mobile phase were directly injected and peak areas were measured. Finally, the recovery was calculated by comparing the peak areas (in terms of the amount found) of the two sets of samples, and recoveries ranged in between 90 and 96% (Table 3). These results suggest the developed method is of high precision and accuracy.

Table 3

Recovery of GSH from PC-12 cells after spiking GSH.

GSH concentration (µg/mL)	Amount found in mobile phase (µg/mL)a	Amount found in PC-12 cells (µg/mL)a	% recovery
20	19.8	17.9	90.4
40	38.9	37.2	95.9
60	59.8	57.4	95.7

aMean of six replicates.

3.2.4. Robustness and Stability

The robustness of a method is tested by making slight deliberate changes to the separation parameters of the developed method. It was evaluated by varying method parameters such as changes in the pH (2.48–2.52), flow rate (0.6–1.0 mL/min), gradient time (18–22 min), HPLC columns (different lots or suppliers), injection volume (48–52 μL), and wavelength (278–282 nm). The samples responded, according to changes (Table 4).

Table 4

Summary of robustness studies.

Experimental conditions	Variation	RT (min) of Peaks 1, 2, and 3	Combined peak areas	Tailing factor
Change in gradient time (min)	18	6.22, 10.22 & 13.25	58992	1.20
	20 (nominal)	7.52, 11.02 & 14.18	55002	1.20
	22	8.29, 11.89 & 14.98	54999	1.21
Buffer pH	2.48	7.51, 11.12 & 14.21	55022	1.22
	2.5 (nominal)	7.52, 11.02 & 14.19	55002	1.20
	2.52	7.52, 11.08, & 14.17	55462	1.22
Wavelength (nm)	282	7.50, 11.06 & 14.28	56734	1.23
	280 (nominal)	7.52, 11.02, 14.35	55002	1.20
	278	7.55, 11.07, 14.44	53878	1.20
Flow rate (mL/min)	0.6	10.35, 14.22 & 17.85	55474	1.18
	0.8 (nominal)	7.52, 11.02 & 14.44	55002	1.20
	1.0	5.6, 8.62 & 11.28	54878	1.23
Injection volume (µL)	48	7.49, 10.99 & 14.84	53998	1.21
	50 (nominal)	7.52, 11.02 & 14.47	55002	1.20
	52	7.50, 11.01 & 14.25	56878	1.21
Column type	Zorbax C8	7.66, 11.32 & 14.24	52345	1.25
	Eclipse XDB C 8	7.52, 11.02 & 14.44	55002	1.20
	Zorbax C18	7.92, 11.92 & 14.65	60233	1.49

Note: RT: retention time; Peak 1 is 2-nitro-5-mercapto-benzoic acid, Peak 2 is glutathione dimer, and Peak 3 is Ellman's reagent.

Stability of the prepared standard solution was monitored from 0 to 6 h; peak areas and RT were checked against freshly prepared solutions. The results (Figure 5) are expressed in terms of percentage change in peak area. For the stability study in PC-12 cells, samples were spiked with GSH (100 μg/mL) and the stability was accessed from 0 to 6 h, and also samples stored under 4°C were analyzed. From these results the sample seems to be less stable, so throughout the studies freshly prepared samples were used to study the validation parameters.

Figure 5

Stability studies of glutathione over a time period of 6 h.

3.2.5. Biomedical Application

Glutathione is required for the detoxification of methylglyoxal, a toxic metabolite of liver. It is evident from earlier reports that methylglyoxal accumulated in cells due to GSH depletion is the major cause for cellular dysfunction and oxidative stress [17]. Such stress is also observed in conditions such as inflammation, and the cell copes with the stress with intracellular antioxidants. GSH is a major antioxidant present in cells and it exists in reduced as well as oxidized form, depending upon the oxidative state of the cell. An accurate measurement of intracellular GSH provides a means of determining the oxidative stress caused by an agent and the cellular response to it. Both methylglyoxal (Figure 3(b)) and CoCl2 (Figure 3(c)) treatment significantly altered the level of intracellular GSH compared to control (Figure 3(a)). It is possible that CoCl2 also activates GSH synthesizing enzymes, resulting in a higher level of GSH. Further studies will need to be done to test this possibility. There is a previous report that methylglyoxal interferes with GSH synthesis and secretion [18], which could be why methylglyoxal treated cells show a lower level of GSH, compared to control and CoCl2 treated cells. The present study was performed twice with the same sample procedure and HPLC chromatographic conditions. When compared with the control, in both the studies authors found a significant decrease in GSH upon methylglyoxal treatment. Further experimentation is needed to study the more accurate and precise results.

Figure 3

Representative chromatogram from 6 replicates is shown. (a) 2-Nitro-5-mercapto-benzoic (NMB) acid, Peak 1 with RT 7.43 min; glutathione dimer (GSH Dimer), Peak 2 with RT 10.93 min, and Ellman's reagent (DTNB), Peak 3 RT 14.12 min in untreated PC-12 cells (control). (b) NMB, Peak 1 with RT 7.44 min, GSH Dimer, Peak 2 with RT 10.88 min, and DTNB, Peak 3 retention time 14.10 min in PC-12 cells treated with methylglyoxal. (c) NMB, Peak 1 with RT 7.59 min, glutathione dimer, Peak 2 with RT 10.92 min, and DTNB, Peak 3 RT 14.44 min in PC-12 cells treated with CoCl2.

4. Conclusion

A simple, easy, and reliable LC method was developed and validated as per the standard guidelines for the determination of GSH in PC-12 cells. A gradient time of 20 min, with a segmented gradient range and flow rate, was found optimum when a C8 column was used. The validation parameters showed satisfactory linearity in the range of 1–20 μg/mL with a high degree of precision and accuracy. To the best of our knowledge this is the first report on measurement of GSH in PC-12 cells upon treatment with these reagents; furthermore, the assay leads its applicability to study the role of GSH in preventing cellular stress.