RetractionRetracted A Simple HPLC-UV Method for the Determination ofGlutathione in PC-12 Cells

Scientifica

Received 1 February 2022 Accepted 1 February 2022 Published 24 February 2022

Copyright copy 2022 is is an open access article distributed under the Creative Commons Attribution License which permitsunrestricted use distribution and reproduction in any medium provided the original work is properly cited

Scientica has retracted the article titled ldquoA Simple HPLC-UV Method for the Determination of Glutathione in PC-12Cellsrdquo [1] due to concerns with data permissions e listedauthors did not collect the data presented in the article anddid not possess the necessary permissions for publication ofthe data It was found that the data were collected and ownedby Ganesh K Sittampalli and colleagues and the article istherefore retracted from the journal with the agreement ofthe editorial board e authors agree to the retraction

References

[1] R N Appala S Chigurupati R V V S S Appala K KrishnanSelvarajan and J I Mohammad ldquoA Simple HPLC-UVMethodfor the Determination of Glutathione in PC-12 Cellsrdquo Scien-tica vol 2016 Article ID 6897890 6 pages 2016

HindawiScientificaVolume 2022 Article ID 9781919 1 pagehttpsdoiorg10115520229781919

RETRACTEDResearch Article

A Simple HPLC-UV Method for the Determination ofGlutathione in PC-12 Cells

Raju N Appala1 Sridevi Chigurupati2 Raju V V S S Appala3

Kesavanarayanan Krishnan Selvarajan4 and Jahidul IslamMohammad5

1Department of Pharmaceutical Chemistry Sultan Ul Uloom College of Pharmacy Telangana Hyderabad 500 034 India2Department of Pharmaceutical Chemistry Faculty of Pharmacy AIMST University Semeling 08100 Bedong Kedah Malaysia3Department of Chemistry Faculty of Pharmacy MAHSA University 59100 Kuala Lumpur Malaysia4Faculty of Pharmacy Universiti Teknologi MARA (UiTM) 42300 Puncak Alam Selangor Malaysia5Faculty of Medicine AIMST University Semeling 08100 Bedong Kedah Malaysia

Correspondence should be addressed to Sridevi Chigurupati srideviphdgmailcom

Received 18 December 2015 Revised 6 March 2016 Accepted 7 March 2016

Academic Editor Qian Wang

Copyright copy 2016 Raju N Appala et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

A highly sensitive and simple HPLC-UV method was developed and validated for the assay of glutathione (GSH) in PC-12 cellsGlutathione is a major intracellular antioxidant having multiple biological effects best known for its cytoprotective effects againstcell damage from reactive oxygen species and toxic reactive metabolites and regulating the cellular redox homeostasis Due to itsown sulfhydryl (SH) group GSH readily reacts with Ellmanrsquos reagent to form a stable dimer which allows for quantitative estimationof GSH in biological systems by UV detection The separation was achieved using a C

8column with a mobile phase consisting of

phosphate buffer adjusted to pH 25 (mobile phase A) and acetonitrile (mobile phase B) running in a segmented gradientmanner ata flow rate of 08mLmin andUVdetectionwas performed at 280 nmThedevelopedHPLC-UVmethodwas validatedwith respectto precision accuracy robustness and linearity within a range of 1ndash20 120583gmL Limit of detection (LOD) and limit of quantification(LOQ) were 005 and 01 120583gmL respectively Furthermore the method shows the applicability for monitoring the oxidative stressin PC-12 cells

1 Introduction

Glutathione (GSH) is chemically known as (2S)-2-amino-4-[[(1R)-[(carboxymethyl) carbamoyl]-2-sulfanylethyl] car-bamoyl] butanoic acid GSH is a tripeptide (Figure 1) oftenconsidered as the mother of all antioxidants and is presentin almost every cell Because GSH exists within the cellsit is in a prime position to neutralize free radicals Thestrong antioxidant effect of GSH helps keep cells runningsmoothly and also helps the liver to remove chemicals thatare foreign to the body such as drugspollutants [1 2] Inaddition GSH has the potential to fight almost any diseaseparticularly those associated with ageing since free radicaldamage is the cause of many of the diseases of old age GSH isnucleophilic at the sulfur and attacks poisonous electrophilicconjugate acceptors Thiol groups are kept in a reduced stateat a concentration of approximately sim5mM in animal cells

In effect GSH reduces any disulfide bond formed withincytoplasmic proteins to cysteines by acting as an electrondonor In the process GSH is converted to its oxidized formglutathione disulfide (GSSG) Glutathione is found almostexclusively in its reduced form since the enzyme that revertsit from its oxidized form GSSG is constitutively active andinducible upon oxidative stress In fact the ratio of GSH toGSSG within cells is often used scientifically as a measure ofcellular toxicity [3ndash5]

In healthy cells and tissue more than 90 of the totalglutathione pool is in the reduced form and less than 10exists in the disulfide form [6] An increased GSSG-to-GSH ratio is considered indicative of oxidative stress Severalmethodswere reported earlier to estimate the amount of GSHpresent in biological samples and commercial products usingHPLC [7 8] capillary zone electrophoresis [9ndash11] However asimpleHPLC-UVmethod for quantification of GSH in PC-12

Hindawi Publishing CorporationScientificaVolume 2016 Article ID 6897890 6 pageshttpdxdoiorg10115520166897890

RETRACTED

2 Scientifica

HN N

H

O

OO

O

HN

NH

O

OO

O

SS

SS

+ +

Glutathione (GSH) Ellmanrsquos reagent (DTNB) Glutathione dimer (GSH Dimer)

OH

NH2

NH2

NO2

NO2

NO2

NO2

OH

HOOC

HOOC

SH

COOH

OH

COOH

OH

Peak 3 Peak 1 Peak 2

HS

2-Nitro-5-mercapto-benzoic (NMB) acid

Figure 1 Reaction of Ellmanrsquos reagent with glutathione

cells and its role in cellular stress is yet to be foundThis studyattempts to develop and validate a simple HPLC-UVmethodfor the determination of GSH in PC-12 cells

2 Materials and Method

21 Chemicals and Reagents Glutathione (GSH-reducedform) was purchased from Acros (USA) 551015840-dithio-bis(2-nitrobenzoic acid) (DTNB or Ellmanrsquos reagent) DulbeccorsquosModified Eagle Medium (DMEM) Fetal Bovine Serum(FBS) acetonitrile (ACN) and potassium monobasic phos-phate were obtained from Fisher Scientific Co and What-man Grade 1 Qualitative Filtration Paper phosphoric acidand tri-chloroacetic acid (TCA) were obtained from Sigma-Aldrich (USA) In-house purified deionized (DI) water wasused throughout the investigation

22 HPLC Instrument and Separation Parameters HitachiLaChrom series LC system consisting of an L-7100 pumpan L-7200 autosampler an L-7400 UV detector set at awavelength of 280 nm and D-7000 interface with systemmanager data acquisition software (version 50) was usedthroughout the study The chromatographic separation wasachieved using anAgilent Eclipse XDBC

8(150times 46mm 5120583)

column the optimized method used a segmented gradientmobile phase with phosphate buffer at pH = 25 as solvent (A)and ACN as solvent (B) and the gradient program is shownin Table 1The sample volume for injection was 50 120583L and thetotal run time was 20min

23 Preparation of Standard Solution A stock solution ofGSH at 100 120583gmL concentrations was prepared by weighing10mg of GSH in 100mL volumetric flask and making up thevolume with DI water The stock solution was stored at 4∘Cand appropriate dilutions of GSH were prepared to makeworking standards of 01 05 1 2 5 10 and 20 120583gmL ofvarious validation studies 500120583gmL of Ellmanrsquos reagent wasprepared by accurately weighing 50mg of reagent in 100mLof methanol and stored at 4∘C

Table 1 Summary of gradient program

Time (min) mobile phase(A) Phosphate

buffer

mobile phase(B) ACN

Flow rate(mLmin)

0 90 10 0845 60 40 0846 60 40 05140 60 40 05141 60 40 08200 90 90 08

24 Sample Preparation and Withdrawal of GSH from PC-12Cells Cells (1 times 106 cellssample) were cultured in DMEMsupplemented with 10 FBS at 37∘C for 24 h followed bytreatment with eitherMnCl

2(5mM) CoCl

2(5mM)methyl-

glyoxal (04mM) or hydrogen peroxide (01) for additional24 h Cells grown in DMEM alone were used as controlsCells were centrifuged at 3000 rpm for 90 seconds and thesupernatant was discarded The cell pellet was suspended in10 ice-cold TCA and centrifuged for 15min at 9000timesgThesupernatant was collected and GSH was measured by usingHPLC-UV

25 Derivatization of GSH Since its introduction in 1959Ellmanrsquos reagent has been the favorite reagent for spectropho-tometric measurement of protein sulfhydryls For GSH anal-ysis an aliquot of 05mL of GSH solution was added to05mL of 05mM Ellmanrsquos reagent solutionThe solution wasallowed to react for 30min at 60∘C followed by injecting intothe HPLC at a flow rate of 08mLmin the separation wasperformed at ambient temperature and the detection wascarried at 280 nm For biological samples the sample fromPC-12 cells was first filtered through Millipore membranefollowed by the addition of 05mL Ellmanrsquos reagent (05mM)

3 Results and Discussion

31 Method Development From the structure of GSH it isvery clear that the compound is highly polar however the

RETRACTED

Scientifica 3

1483

1123

765

DTNBNMB

GSHDimer

050

100150200250300

Inte

nsity

(mV

)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 180Retention time (min)

1483

1123

765

DTNBNMB

GSHDimer

Figure 2 Representative chromatogram from 6 replicates is shown2-Nitro-5-mercapto-benzoic (NMB) acid glutathione dimer (GSHDimer) and Ellmanrsquos reagent (DTNB) in GSH raw material

compound was found to be insoluble in ACN and methanoland completely soluble in water In the development of a RP-HPLC method for GSH it was determined that GSH is notretained in the RP columns therefore we choose to derivatizethe sample with Ellmanrsquos reagent popularly used to quantifythiols [12] The proposed derivatized compounds (Figure 1)are 2-nitro-5-mercapto-benzoic (NMB) acid and glutathionedimer (GSH Dimer) These compounds have demonstratedsome increase in hydrophobicity andwere effectively retainedin the RP columns allowing the separation of compounds inthe sample to occur Evidence has shown that the maximumUV-Vis spectrum absorbance for the dimer is at 412 nm Forthis experiment an appropriate wavelength was selected at280 nm to reduce the baseline disturbances and improve thesignal strength Next step was to optimize the separationconditions different RP columns were tested and finallyAgilent Eclipse XDB C

8column was selected The mobile

phase consisted of phosphate buffer at pH = 25 as solvent(A) and ACN as solvent (B) the main reason to select a pHat 25 was to protonate all the free silanols in the column andto reduce their chromatographic activity [13]

A segmented gradient program (Table 1) was used toachieve separation with the retention times (RT) of NMBGSH Dimer and DTNB at 765 1123 and 1483 respectively(Figure 2) When the developed method was tested on sam-ples extracted from PC-12 cells no endogenous interferingpeaks were observed in the individual blank sample at theRT of GSH biosample making the developedmethod of highruggedness

32 Validation of Developed Method Method validation wasperformed in terms of system suitability linearity precisionaccuracy robustness and finally sensitivity [14ndash16]

321 System Suitability System suitability tests (SST) arean integral part of liquid chromatography methods Theyare used to verify that the resolution and reproducibilityof the chromatography system are adequate for the analysisto be done The system suitability test is a US Food andDrug Administration (FDA) validation requirement [14ndash16]and is usually considered as a prevalidation requirement(equipment performance qualification test) SST was evalu-ated by injecting 3 blank samples (diluting solvent) followed

Table 2 Intraday and interday precision studies for the determina-tion of GSH using HPLC-UV

Added (120583gmL)Intraday

Found (120583gmL)plusmn SD

RSD(119899 = 6) Recovery ()

20 197 13 9840 401 22 1002560 588 19 98

Interday (119899 = 6)20 192 18 9640 392 15 9860 572 27 953

by 6 injections of GSH (100 120583gmL) Parameters such asUSP plate count were found to be 5878 tailing factor is 11resolution is 242 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 forthe method The RSD was found to be less than 10

322 Linearity and Sensitivity Calibration curves wereobtained (using least squares method) by plotting the con-centration ratio versus the peak area ratio for the analyte(Figure 4)Themethod showed linearity within the range of 1to 20 120583gmL with a correlation cost ability greater than 0998The LOD was defined as the compound concentration thatproduces a signal-to-noise (119878119873) ratio greater than three andit was found to be 005 120583gmL The limit of quantitation forthe assay was evaluated as the concentration ten times to 119878119873ratio and was found to be 01 120583gmL

323 Precision and Recovery Injection precision (repeatabil-ity) was determined by six injections of standard and also bycalculating the system suitability factors The method preci-sionwas carried out by freshly prepared standards andRSDvalue was calculated for peak areas For examining interdayprecision (reproducibility) the samples were analyzed bysecond chemist on a different day using a different instrumentwith help of freshly prepared samples Satisfactory repeata-bility and precision were achieved with RSD values withinthe limitsThe acceptance criterion for repeatability (intradayprecision) and intermediate (interday precision) RSD shouldbe better than 20 at lower concentrations and better than15 at higher concentrations (Table 2)

The recovery of GSH from PC-12 cells was estimated byspiking 20 40 and 60 120583gmL concentration in six replicatesSix replicate samples containing the same strength of GSHin mobile phase were directly injected and peak areas weremeasured Finally the recovery was calculated by comparingthe peak areas (in terms of the amount found) of the two setsof samples and recoveries ranged in between 90 and 96(Table 3) These results suggest the developed method is ofhigh precision and accuracy

324 Robustness and Stability The robustness of a method istested by making slight deliberate changes to the separation

RETRACTED

4 Scientifica

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 180Retention time (min)

0

5

10

15

20

25

30In

tens

ity (m

V)

1412

1093743NMB

DTNB

GSHDimer

1412

1093743NMB

DTNB

GSHDimer

(a)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 180Retention time (min)

1410

1088744NMB

DTNB

GSHDimer

0

5

10

15

20

25

30

Inte

nsity

(mV

)

1410

1088744NMB

DTNB

GSHDimmD er

(b)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 180Retention time (min)

05

10152025303540455055

Inte

nsity

(mV

)

1444

1092

759

NMB

GSHDimer

1444

1092

759

NMBB

GSHDimerr

DTNB

(c)

Figure 3 Representative chromatogram from 6 replicates is shown (a) 2-Nitro-5-mercapto-benzoic (NMB) acid Peak 1 with RT 743minglutathione dimer (GSH Dimer) Peak 2 with RT 1093min and Ellmanrsquos reagent (DTNB) Peak 3 RT 1412min in untreated PC-12 cells(control) (b) NMB Peak 1 with RT 744min GSH Dimer Peak 2 with RT 1088min and DTNB Peak 3 retention time 1410min in PC-12cells treated with methylglyoxal (c) NMB Peak 1 with RT 759min glutathione dimer Peak 2 with RT 1092min and DTNB Peak 3 RT1444min in PC-12 cells treated with CoCl

2

NMBGSH Dimer

0

200

400

600

800

1000

1200

1400

1600

Resp

onse

fact

or (v

olt)

5 10 15 20 250Concentration

y = 6620x minus 2142

R2 = 0999

R2 = 0999y = 33x minus 1

Figure 4 Linearity plot of 2-nitro-5-mercapto-benzoic (NMB) acidand glutathione dimer (GSH Dimer)

parameters of the developed method It was evaluated byvarying method parameters such as changes in the pH (248ndash252) flow rate (06ndash10mLmin) gradient time (18ndash22min)

Table 3 Recovery of GSH from PC-12 cells after spiking GSH

GSHconcentration(120583gmL)

Amount foundin mobile phase

(120583gmL)a

Amount foundin PC-12 cells(120583gmL)a

recovery

20 198 179 90440 389 372 95960 598 574 957aMean of six replicates

HPLC columns (different lots or suppliers) injection vol-ume (48ndash52120583L) and wavelength (278ndash282 nm)The samplesresponded according to changes (Table 4)

Stability of the prepared standard solution wasmonitoredfrom 0 to 6 h peak areas and RT were checked against freshlyprepared solutions The results (Figure 5) are expressed interms of percentage change in peak area For the stabil-ity study in PC-12 cells samples were spiked with GSH(100 120583gmL) and the stability was accessed from 0 to 6 h andalso samples stored under 4∘C were analyzed From theseresults the sample seems to be less stable so throughoutthe studies freshly prepared samples were used to study thevalidation parameters

RETRACTED

Scientifica 5

Table 4 Summary of robustness studies

Experimental conditions Variation RT (min) ofPeaks 1 2 and 3

Combined peakareas Tailing factor

Change in gradient time(min)

18 622 1022 amp 1325 58992 12020 (nominal) 752 1102 amp 1418 55002 120

22 829 1189 amp 1498 54999 121

Buffer pH

248 751 1112 amp 1421 55022 12225 (nominal) 752 1102 amp 1419 55002 120

252 752 1108 amp 1417 55462 122

Wavelength (nm)282 750 1106 amp 1428 56734 123

280 (nominal) 752 1102 1435 55002 120278 755 1107 1444 53878 120

Flow rate (mLmin)

06 1035 1422 amp 1785 55474 11808 (nominal) 752 1102 amp 1444 55002 120

10 56 862 amp 1128 54878 123

Injection volume (120583L)

48 749 1099 amp 1484 53998 12150 (nominal) 752 1102 amp 1447 55002 120

52 750 1101 amp 1425 56878 121

Column type

Zorbax C8 766 1132 amp 1424 52345 125

Eclipse XDB C8 752 1102 amp 1444 55002 120Zorbax C

18 792 1192 amp 1465 60233 149Note RT retention time Peak 1 is 2-nitro-5-mercapto-benzoic acid Peak 2 is glutathione dimer and Peak 3 is Ellmanrsquos reagent

0

50

100

150

200

250

300

350

400

450

500

Peak

area

(vol

ts)

50 100 150 200 250 300 3500Time (minutes)

GSH (raw)GSH in PC-12 cells

Figure 5 Stability studies of glutathione over a time period of 6 h

325 Biomedical Application Glutathione is required for thedetoxification ofmethylglyoxal a toxicmetabolite of liver It isevident from earlier reports that methylglyoxal accumulatedin cells due to GSH depletion is the major cause for cellulardysfunction and oxidative stress [17] Such stress is alsoobserved in conditions such as inflammation and the cellcopes with the stress with intracellular antioxidants GSH isa major antioxidant present in cells and it exists in reduced

as well as oxidized form depending upon the oxidative stateof the cell An accurate measurement of intracellular GSHprovides a means of determining the oxidative stress causedby an agent and the cellular response to it Bothmethylglyoxal(Figure 3(b)) and CoCl

2(Figure 3(c)) treatment significantly

altered the level of intracellular GSH compared to control(Figure 3(a)) It is possible that CoCl

2also activates GSH

synthesizing enzymes resulting in a higher level of GSHFurther studies will need to be done to test this possibilityThere is a previous report that methylglyoxal interferes withGSH synthesis and secretion [18] which could be whymethylglyoxal treated cells show a lower level of GSH com-pared to control and CoCl

2treated cells The present study

was performed twice with the same sample procedure andHPLC chromatographic conditions When compared withthe control in both the studies authors found a significantdecrease in GSH upon methylglyoxal treatment Furtherexperimentation is needed to study the more accurate andprecise results

4 Conclusion

A simple easy and reliable LC method was developed andvalidated as per the standard guidelines for the determinationof GSH in PC-12 cells A gradient time of 20min with asegmented gradient range and flow rate was found optimumwhen a C

8column was used The validation parameters

showed satisfactory linearity in the range of 1ndash20120583gmL witha high degree of precision and accuracy To the best of ourknowledge this is the first report on measurement of GSH inPC-12 cells upon treatment with these reagents furthermore

RETRACTED

6 Scientifica

the assay leads its applicability to study the role of GSH inpreventing cellular stress

Competing Interests

The authors declare that they have no competing interests

References

[1] N Gadoth and H H Gobel Oxidative Stress and Free RadicalDamage in Neurology Humana Press 2010

[2] B Sharma S Singh and N J Siddiqi ldquoBiomedical implicationsof heavy metals induced imbalances in redox systemsrdquo BioMedResearch International vol 2014 Article ID 640754 26 pages2014

[3] J E Ash S Budavari M OrsquoNeill A Smith P E Heckelmanand J KinnearyTheMerck Index Chapman amp Hall New YorkNY USA 11th edition 1996

[4] A Pompella A Visvikis A Paolicchi V De Tata and A FCasini ldquoThe changing faces of glutathione a cellular protago-nistrdquo Biochemical Pharmacology vol 66 no 8 pp 1499ndash15032003

[5] A Pastore F Piemonte M Locatelli et al ldquoDeterminationof blood total reduced and oxidized glutathione in pediatricsubjectsrdquo Clinical Chemistry vol 47 no 8 pp 1467ndash1469 2001

[6] M C Reed R L Thomas J Pavisic S J James C MUlrich andH FNijhout ldquoAmathematicalmodel of glutathionemetabolismrdquo Theoretical Biology and Medical Modelling vol 5pp 8ndash10 2008

[7] T Santa ldquoRecent advances in analysis of glutathione in bio-logical samples by high-performance liquid chromatography abrief overviewrdquo Drug Discoveries and Therapeutics vol 7 no 5pp 172ndash177 2013

[8] L-P Yap H Sancheti M D Ybanez J Garcia E Cadenas andDHan ldquoDetermination ofGSHGSSG andGSNOusingHPLCwith electrochemical detectionrdquo Methods in Enzymology vol473 pp 137ndash147 2010

[9] Z D Zhou and T M Lim ldquoRoles of glutathione (GSH) indopamine (DA) oxidation studied by improved tandem HPLCplus ESI-MSrdquo Neurochemical Research vol 34 no 2 pp 316ndash326 2009

[10] M Yan G-B Shi Y Sui T Guo J-W Zhang and S-J FanldquoDetermination of reduced glutathione in human plasma byRP-HPLCrdquo Pharmaceutical Journal of Chinese Peoplersquos Libera-tion Army vol 3 pp 251ndash253 2008

[11] P Zhu T Oe and I A Blair ldquoDetermination of cellular redoxstatus by stable isotope dilution liquid chromatographymassspectrometry analysis of glutathione and glutathione disulfiderdquoRapid Communications in Mass Spectrometry vol 22 no 4 pp432ndash440 2008

[12] L Bergstrom ldquoSome pathologies of sensory and neural hearinglossrdquo Canadian Journal of Otolaryngology Supplement vol 2pp 1ndash28 1975

[13] L R Snyder J J Kirkland and J L Glajch Practical HPLCMethod Development John Wiley amp Sons Hoboken NJ USA1997

[14] ldquoQ2b validation of analytical procedures methodologyrdquo inProceedings of the International Conference on Harmonization(ICH rsquo97) p 27463 US FDA Federal Register May 1997

[15] G A Shabir ldquoValidation of high-performance liquid chro-matography methods for pharmaceutical analysis understand-ing the differences and similarities between validation require-ments of the US Food and Drug Administration the USPharmacopeia and the International Conference on Harmo-nizationrdquo Journal of Chromatography A vol 987 no 1-2 pp 57ndash66 2003

[16] M Bakshi and S Singh ldquoDevelopment of validated stability-indicating assay methodsmdashcritical reviewrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 28 no 6 pp 1011ndash10402002

[17] A Riboulet-Chavey A Pierron I Durand J Murdaca JGiudicelli and E Van Obberghen ldquoMethylglyoxal impairs theinsulin signaling pathways independently of the formation ofintracellular reactive oxygen speciesrdquoDiabetes vol 55 no 5 pp1289ndash1299 2006

[18] Y-D Hsuuw C-K Chang W-H Chan and J-S Yu ldquoCur-cumin prevents methylglyoxal-induced oxidative stress andapoptosis in mouse embryonic stem cells and blastocystsrdquoJournal of Cellular Physiology vol 205 no 3 pp 379ndash386 2005

RETRACTED

Scientifica 3

1483

1123

765

DTNBNMB

GSHDimer

050

100150200250300

Inte

nsity

(mV

)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 180Retention time (min)

1483

1123

765

DTNBNMB

GSHDimer

Figure 2 Representative chromatogram from 6 replicates is shown2-Nitro-5-mercapto-benzoic (NMB) acid glutathione dimer (GSHDimer) and Ellmanrsquos reagent (DTNB) in GSH raw material

compound was found to be insoluble in ACN and methanoland completely soluble in water In the development of a RP-HPLC method for GSH it was determined that GSH is notretained in the RP columns therefore we choose to derivatizethe sample with Ellmanrsquos reagent popularly used to quantifythiols [12] The proposed derivatized compounds (Figure 1)are 2-nitro-5-mercapto-benzoic (NMB) acid and glutathionedimer (GSH Dimer) These compounds have demonstratedsome increase in hydrophobicity andwere effectively retainedin the RP columns allowing the separation of compounds inthe sample to occur Evidence has shown that the maximumUV-Vis spectrum absorbance for the dimer is at 412 nm Forthis experiment an appropriate wavelength was selected at280 nm to reduce the baseline disturbances and improve thesignal strength Next step was to optimize the separationconditions different RP columns were tested and finallyAgilent Eclipse XDB C

8column was selected The mobile

phase consisted of phosphate buffer at pH = 25 as solvent(A) and ACN as solvent (B) the main reason to select a pHat 25 was to protonate all the free silanols in the column andto reduce their chromatographic activity [13]

A segmented gradient program (Table 1) was used toachieve separation with the retention times (RT) of NMBGSH Dimer and DTNB at 765 1123 and 1483 respectively(Figure 2) When the developed method was tested on sam-ples extracted from PC-12 cells no endogenous interferingpeaks were observed in the individual blank sample at theRT of GSH biosample making the developedmethod of highruggedness

32 Validation of Developed Method Method validation wasperformed in terms of system suitability linearity precisionaccuracy robustness and finally sensitivity [14ndash16]

321 System Suitability System suitability tests (SST) arean integral part of liquid chromatography methods Theyare used to verify that the resolution and reproducibilityof the chromatography system are adequate for the analysisto be done The system suitability test is a US Food andDrug Administration (FDA) validation requirement [14ndash16]and is usually considered as a prevalidation requirement(equipment performance qualification test) SST was evalu-ated by injecting 3 blank samples (diluting solvent) followed

Table 2 Intraday and interday precision studies for the determina-tion of GSH using HPLC-UV

Added (120583gmL)Intraday

Found (120583gmL)plusmn SD

RSD(119899 = 6) Recovery ()

20 197 13 9840 401 22 1002560 588 19 98

Interday (119899 = 6)20 192 18 9640 392 15 9860 572 27 953

by 6 injections of GSH (100 120583gmL) Parameters such asUSP plate count were found to be 5878 tailing factor is 11resolution is 242 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 forthe method The RSD was found to be less than 10

322 Linearity and Sensitivity Calibration curves wereobtained (using least squares method) by plotting the con-centration ratio versus the peak area ratio for the analyte(Figure 4)Themethod showed linearity within the range of 1to 20 120583gmL with a correlation cost ability greater than 0998The LOD was defined as the compound concentration thatproduces a signal-to-noise (119878119873) ratio greater than three andit was found to be 005 120583gmL The limit of quantitation forthe assay was evaluated as the concentration ten times to 119878119873ratio and was found to be 01 120583gmL

323 Precision and Recovery Injection precision (repeatabil-ity) was determined by six injections of standard and also bycalculating the system suitability factors The method preci-sionwas carried out by freshly prepared standards andRSDvalue was calculated for peak areas For examining interdayprecision (reproducibility) the samples were analyzed bysecond chemist on a different day using a different instrumentwith help of freshly prepared samples Satisfactory repeata-bility and precision were achieved with RSD values withinthe limitsThe acceptance criterion for repeatability (intradayprecision) and intermediate (interday precision) RSD shouldbe better than 20 at lower concentrations and better than15 at higher concentrations (Table 2)

The recovery of GSH from PC-12 cells was estimated byspiking 20 40 and 60 120583gmL concentration in six replicatesSix replicate samples containing the same strength of GSHin mobile phase were directly injected and peak areas weremeasured Finally the recovery was calculated by comparingthe peak areas (in terms of the amount found) of the two setsof samples and recoveries ranged in between 90 and 96(Table 3) These results suggest the developed method is ofhigh precision and accuracy

324 Robustness and Stability The robustness of a method istested by making slight deliberate changes to the separation

RETRACTED

4 Scientifica

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 180Retention time (min)

0

5

10

15

20

25

30In

tens

ity (m

V)

1412

1093743NMB

DTNB

GSHDimer

1412

1093743NMB

DTNB

GSHDimer

(a)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 180Retention time (min)

1410

1088744NMB

DTNB

GSHDimer

0

5

10

15

20

25

30

Inte

nsity

(mV

)

1410

1088744NMB

DTNB

GSHDimmD er

(b)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 180Retention time (min)

05

10152025303540455055

Inte

nsity

(mV

)

1444

1092

759

NMB

GSHDimer

1444

1092

759

NMBB

GSHDimerr

DTNB

(c)

Figure 3 Representative chromatogram from 6 replicates is shown (a) 2-Nitro-5-mercapto-benzoic (NMB) acid Peak 1 with RT 743minglutathione dimer (GSH Dimer) Peak 2 with RT 1093min and Ellmanrsquos reagent (DTNB) Peak 3 RT 1412min in untreated PC-12 cells(control) (b) NMB Peak 1 with RT 744min GSH Dimer Peak 2 with RT 1088min and DTNB Peak 3 retention time 1410min in PC-12cells treated with methylglyoxal (c) NMB Peak 1 with RT 759min glutathione dimer Peak 2 with RT 1092min and DTNB Peak 3 RT1444min in PC-12 cells treated with CoCl

2

NMBGSH Dimer

0

200

400

600

800

1000

1200

1400

1600

Resp

onse

fact

or (v

olt)

5 10 15 20 250Concentration

y = 6620x minus 2142

R2 = 0999

R2 = 0999y = 33x minus 1

Figure 4 Linearity plot of 2-nitro-5-mercapto-benzoic (NMB) acidand glutathione dimer (GSH Dimer)

parameters of the developed method It was evaluated byvarying method parameters such as changes in the pH (248ndash252) flow rate (06ndash10mLmin) gradient time (18ndash22min)

Table 3 Recovery of GSH from PC-12 cells after spiking GSH

GSHconcentration(120583gmL)

Amount foundin mobile phase

(120583gmL)a

Amount foundin PC-12 cells(120583gmL)a

recovery

20 198 179 90440 389 372 95960 598 574 957aMean of six replicates

HPLC columns (different lots or suppliers) injection vol-ume (48ndash52120583L) and wavelength (278ndash282 nm)The samplesresponded according to changes (Table 4)

Stability of the prepared standard solution wasmonitoredfrom 0 to 6 h peak areas and RT were checked against freshlyprepared solutions The results (Figure 5) are expressed interms of percentage change in peak area For the stabil-ity study in PC-12 cells samples were spiked with GSH(100 120583gmL) and the stability was accessed from 0 to 6 h andalso samples stored under 4∘C were analyzed From theseresults the sample seems to be less stable so throughoutthe studies freshly prepared samples were used to study thevalidation parameters

RETRACTED

Scientifica 5

Table 4 Summary of robustness studies

Experimental conditions Variation RT (min) ofPeaks 1 2 and 3

Combined peakareas Tailing factor

Change in gradient time(min)

18 622 1022 amp 1325 58992 12020 (nominal) 752 1102 amp 1418 55002 120

22 829 1189 amp 1498 54999 121

Buffer pH

248 751 1112 amp 1421 55022 12225 (nominal) 752 1102 amp 1419 55002 120

252 752 1108 amp 1417 55462 122

Wavelength (nm)282 750 1106 amp 1428 56734 123

280 (nominal) 752 1102 1435 55002 120278 755 1107 1444 53878 120

Flow rate (mLmin)

06 1035 1422 amp 1785 55474 11808 (nominal) 752 1102 amp 1444 55002 120

10 56 862 amp 1128 54878 123

Injection volume (120583L)

48 749 1099 amp 1484 53998 12150 (nominal) 752 1102 amp 1447 55002 120

52 750 1101 amp 1425 56878 121

Column type

Zorbax C8 766 1132 amp 1424 52345 125

Eclipse XDB C8 752 1102 amp 1444 55002 120Zorbax C

18 792 1192 amp 1465 60233 149Note RT retention time Peak 1 is 2-nitro-5-mercapto-benzoic acid Peak 2 is glutathione dimer and Peak 3 is Ellmanrsquos reagent

0

50

100

150

200

250

300

350

400

450

500

Peak

area

(vol

ts)

50 100 150 200 250 300 3500Time (minutes)

GSH (raw)GSH in PC-12 cells

Figure 5 Stability studies of glutathione over a time period of 6 h

325 Biomedical Application Glutathione is required for thedetoxification ofmethylglyoxal a toxicmetabolite of liver It isevident from earlier reports that methylglyoxal accumulatedin cells due to GSH depletion is the major cause for cellulardysfunction and oxidative stress [17] Such stress is alsoobserved in conditions such as inflammation and the cellcopes with the stress with intracellular antioxidants GSH isa major antioxidant present in cells and it exists in reduced

as well as oxidized form depending upon the oxidative stateof the cell An accurate measurement of intracellular GSHprovides a means of determining the oxidative stress causedby an agent and the cellular response to it Bothmethylglyoxal(Figure 3(b)) and CoCl

2(Figure 3(c)) treatment significantly

altered the level of intracellular GSH compared to control(Figure 3(a)) It is possible that CoCl

2also activates GSH

synthesizing enzymes resulting in a higher level of GSHFurther studies will need to be done to test this possibilityThere is a previous report that methylglyoxal interferes withGSH synthesis and secretion [18] which could be whymethylglyoxal treated cells show a lower level of GSH com-pared to control and CoCl

2treated cells The present study

was performed twice with the same sample procedure andHPLC chromatographic conditions When compared withthe control in both the studies authors found a significantdecrease in GSH upon methylglyoxal treatment Furtherexperimentation is needed to study the more accurate andprecise results

4 Conclusion

A simple easy and reliable LC method was developed andvalidated as per the standard guidelines for the determinationof GSH in PC-12 cells A gradient time of 20min with asegmented gradient range and flow rate was found optimumwhen a C

8column was used The validation parameters

showed satisfactory linearity in the range of 1ndash20120583gmL witha high degree of precision and accuracy To the best of ourknowledge this is the first report on measurement of GSH inPC-12 cells upon treatment with these reagents furthermore

RETRACTED

6 Scientifica

the assay leads its applicability to study the role of GSH inpreventing cellular stress

Competing Interests

The authors declare that they have no competing interests

References

[1] N Gadoth and H H Gobel Oxidative Stress and Free RadicalDamage in Neurology Humana Press 2010

[2] B Sharma S Singh and N J Siddiqi ldquoBiomedical implicationsof heavy metals induced imbalances in redox systemsrdquo BioMedResearch International vol 2014 Article ID 640754 26 pages2014

[3] J E Ash S Budavari M OrsquoNeill A Smith P E Heckelmanand J KinnearyTheMerck Index Chapman amp Hall New YorkNY USA 11th edition 1996

[4] A Pompella A Visvikis A Paolicchi V De Tata and A FCasini ldquoThe changing faces of glutathione a cellular protago-nistrdquo Biochemical Pharmacology vol 66 no 8 pp 1499ndash15032003

[5] A Pastore F Piemonte M Locatelli et al ldquoDeterminationof blood total reduced and oxidized glutathione in pediatricsubjectsrdquo Clinical Chemistry vol 47 no 8 pp 1467ndash1469 2001

[6] M C Reed R L Thomas J Pavisic S J James C MUlrich andH FNijhout ldquoAmathematicalmodel of glutathionemetabolismrdquo Theoretical Biology and Medical Modelling vol 5pp 8ndash10 2008

[7] T Santa ldquoRecent advances in analysis of glutathione in bio-logical samples by high-performance liquid chromatography abrief overviewrdquo Drug Discoveries and Therapeutics vol 7 no 5pp 172ndash177 2013

[8] L-P Yap H Sancheti M D Ybanez J Garcia E Cadenas andDHan ldquoDetermination ofGSHGSSG andGSNOusingHPLCwith electrochemical detectionrdquo Methods in Enzymology vol473 pp 137ndash147 2010

[9] Z D Zhou and T M Lim ldquoRoles of glutathione (GSH) indopamine (DA) oxidation studied by improved tandem HPLCplus ESI-MSrdquo Neurochemical Research vol 34 no 2 pp 316ndash326 2009

[10] M Yan G-B Shi Y Sui T Guo J-W Zhang and S-J FanldquoDetermination of reduced glutathione in human plasma byRP-HPLCrdquo Pharmaceutical Journal of Chinese Peoplersquos Libera-tion Army vol 3 pp 251ndash253 2008

[11] P Zhu T Oe and I A Blair ldquoDetermination of cellular redoxstatus by stable isotope dilution liquid chromatographymassspectrometry analysis of glutathione and glutathione disulfiderdquoRapid Communications in Mass Spectrometry vol 22 no 4 pp432ndash440 2008

[12] L Bergstrom ldquoSome pathologies of sensory and neural hearinglossrdquo Canadian Journal of Otolaryngology Supplement vol 2pp 1ndash28 1975

[13] L R Snyder J J Kirkland and J L Glajch Practical HPLCMethod Development John Wiley amp Sons Hoboken NJ USA1997

[14] ldquoQ2b validation of analytical procedures methodologyrdquo inProceedings of the International Conference on Harmonization(ICH rsquo97) p 27463 US FDA Federal Register May 1997

[15] G A Shabir ldquoValidation of high-performance liquid chro-matography methods for pharmaceutical analysis understand-ing the differences and similarities between validation require-ments of the US Food and Drug Administration the USPharmacopeia and the International Conference on Harmo-nizationrdquo Journal of Chromatography A vol 987 no 1-2 pp 57ndash66 2003

[16] M Bakshi and S Singh ldquoDevelopment of validated stability-indicating assay methodsmdashcritical reviewrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 28 no 6 pp 1011ndash10402002

[17] A Riboulet-Chavey A Pierron I Durand J Murdaca JGiudicelli and E Van Obberghen ldquoMethylglyoxal impairs theinsulin signaling pathways independently of the formation ofintracellular reactive oxygen speciesrdquoDiabetes vol 55 no 5 pp1289ndash1299 2006

[18] Y-D Hsuuw C-K Chang W-H Chan and J-S Yu ldquoCur-cumin prevents methylglyoxal-induced oxidative stress andapoptosis in mouse embryonic stem cells and blastocystsrdquoJournal of Cellular Physiology vol 205 no 3 pp 379ndash386 2005

RETRACTED

4 Scientifica

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 180Retention time (min)

0

5

10

15

20

25

30In

tens

ity (m

V)

1412

1093743NMB

DTNB

GSHDimer

1412

1093743NMB

DTNB

GSHDimer

(a)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 180Retention time (min)

1410

1088744NMB

DTNB

GSHDimer

0

5

10

15

20

25

30

Inte

nsity

(mV

)

1410

1088744NMB

DTNB

GSHDimmD er

(b)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 180Retention time (min)

05

10152025303540455055

Inte

nsity

(mV

)

1444

1092

759

NMB

GSHDimer

1444

1092

759

NMBB

GSHDimerr

DTNB

(c)

Figure 3 Representative chromatogram from 6 replicates is shown (a) 2-Nitro-5-mercapto-benzoic (NMB) acid Peak 1 with RT 743minglutathione dimer (GSH Dimer) Peak 2 with RT 1093min and Ellmanrsquos reagent (DTNB) Peak 3 RT 1412min in untreated PC-12 cells(control) (b) NMB Peak 1 with RT 744min GSH Dimer Peak 2 with RT 1088min and DTNB Peak 3 retention time 1410min in PC-12cells treated with methylglyoxal (c) NMB Peak 1 with RT 759min glutathione dimer Peak 2 with RT 1092min and DTNB Peak 3 RT1444min in PC-12 cells treated with CoCl

2

NMBGSH Dimer

0

200

400

600

800

1000

1200

1400

1600

Resp

onse

fact

or (v

olt)

5 10 15 20 250Concentration

y = 6620x minus 2142

R2 = 0999

R2 = 0999y = 33x minus 1

Figure 4 Linearity plot of 2-nitro-5-mercapto-benzoic (NMB) acidand glutathione dimer (GSH Dimer)

parameters of the developed method It was evaluated byvarying method parameters such as changes in the pH (248ndash252) flow rate (06ndash10mLmin) gradient time (18ndash22min)

Table 3 Recovery of GSH from PC-12 cells after spiking GSH

GSHconcentration(120583gmL)

Amount foundin mobile phase

(120583gmL)a

Amount foundin PC-12 cells(120583gmL)a

recovery

20 198 179 90440 389 372 95960 598 574 957aMean of six replicates

HPLC columns (different lots or suppliers) injection vol-ume (48ndash52120583L) and wavelength (278ndash282 nm)The samplesresponded according to changes (Table 4)

Stability of the prepared standard solution wasmonitoredfrom 0 to 6 h peak areas and RT were checked against freshlyprepared solutions The results (Figure 5) are expressed interms of percentage change in peak area For the stabil-ity study in PC-12 cells samples were spiked with GSH(100 120583gmL) and the stability was accessed from 0 to 6 h andalso samples stored under 4∘C were analyzed From theseresults the sample seems to be less stable so throughoutthe studies freshly prepared samples were used to study thevalidation parameters

RETRACTED

Scientifica 5

Table 4 Summary of robustness studies

Experimental conditions Variation RT (min) ofPeaks 1 2 and 3

Combined peakareas Tailing factor

Change in gradient time(min)

18 622 1022 amp 1325 58992 12020 (nominal) 752 1102 amp 1418 55002 120

22 829 1189 amp 1498 54999 121

Buffer pH

248 751 1112 amp 1421 55022 12225 (nominal) 752 1102 amp 1419 55002 120

252 752 1108 amp 1417 55462 122

Wavelength (nm)282 750 1106 amp 1428 56734 123

280 (nominal) 752 1102 1435 55002 120278 755 1107 1444 53878 120

Flow rate (mLmin)

06 1035 1422 amp 1785 55474 11808 (nominal) 752 1102 amp 1444 55002 120

10 56 862 amp 1128 54878 123

Injection volume (120583L)

48 749 1099 amp 1484 53998 12150 (nominal) 752 1102 amp 1447 55002 120

52 750 1101 amp 1425 56878 121

Column type

Zorbax C8 766 1132 amp 1424 52345 125

Eclipse XDB C8 752 1102 amp 1444 55002 120Zorbax C

18 792 1192 amp 1465 60233 149Note RT retention time Peak 1 is 2-nitro-5-mercapto-benzoic acid Peak 2 is glutathione dimer and Peak 3 is Ellmanrsquos reagent

0

50

100

150

200

250

300

350

400

450

500

Peak

area

(vol

ts)

50 100 150 200 250 300 3500Time (minutes)

GSH (raw)GSH in PC-12 cells

Figure 5 Stability studies of glutathione over a time period of 6 h

325 Biomedical Application Glutathione is required for thedetoxification ofmethylglyoxal a toxicmetabolite of liver It isevident from earlier reports that methylglyoxal accumulatedin cells due to GSH depletion is the major cause for cellulardysfunction and oxidative stress [17] Such stress is alsoobserved in conditions such as inflammation and the cellcopes with the stress with intracellular antioxidants GSH isa major antioxidant present in cells and it exists in reduced

as well as oxidized form depending upon the oxidative stateof the cell An accurate measurement of intracellular GSHprovides a means of determining the oxidative stress causedby an agent and the cellular response to it Bothmethylglyoxal(Figure 3(b)) and CoCl

2(Figure 3(c)) treatment significantly

altered the level of intracellular GSH compared to control(Figure 3(a)) It is possible that CoCl

2also activates GSH

synthesizing enzymes resulting in a higher level of GSHFurther studies will need to be done to test this possibilityThere is a previous report that methylglyoxal interferes withGSH synthesis and secretion [18] which could be whymethylglyoxal treated cells show a lower level of GSH com-pared to control and CoCl

2treated cells The present study

was performed twice with the same sample procedure andHPLC chromatographic conditions When compared withthe control in both the studies authors found a significantdecrease in GSH upon methylglyoxal treatment Furtherexperimentation is needed to study the more accurate andprecise results

4 Conclusion

A simple easy and reliable LC method was developed andvalidated as per the standard guidelines for the determinationof GSH in PC-12 cells A gradient time of 20min with asegmented gradient range and flow rate was found optimumwhen a C

8column was used The validation parameters

showed satisfactory linearity in the range of 1ndash20120583gmL witha high degree of precision and accuracy To the best of ourknowledge this is the first report on measurement of GSH inPC-12 cells upon treatment with these reagents furthermore

RETRACTED

6 Scientifica

the assay leads its applicability to study the role of GSH inpreventing cellular stress

Competing Interests

The authors declare that they have no competing interests

References

[1] N Gadoth and H H Gobel Oxidative Stress and Free RadicalDamage in Neurology Humana Press 2010

[2] B Sharma S Singh and N J Siddiqi ldquoBiomedical implicationsof heavy metals induced imbalances in redox systemsrdquo BioMedResearch International vol 2014 Article ID 640754 26 pages2014

[3] J E Ash S Budavari M OrsquoNeill A Smith P E Heckelmanand J KinnearyTheMerck Index Chapman amp Hall New YorkNY USA 11th edition 1996

[4] A Pompella A Visvikis A Paolicchi V De Tata and A FCasini ldquoThe changing faces of glutathione a cellular protago-nistrdquo Biochemical Pharmacology vol 66 no 8 pp 1499ndash15032003

[5] A Pastore F Piemonte M Locatelli et al ldquoDeterminationof blood total reduced and oxidized glutathione in pediatricsubjectsrdquo Clinical Chemistry vol 47 no 8 pp 1467ndash1469 2001

[6] M C Reed R L Thomas J Pavisic S J James C MUlrich andH FNijhout ldquoAmathematicalmodel of glutathionemetabolismrdquo Theoretical Biology and Medical Modelling vol 5pp 8ndash10 2008

[7] T Santa ldquoRecent advances in analysis of glutathione in bio-logical samples by high-performance liquid chromatography abrief overviewrdquo Drug Discoveries and Therapeutics vol 7 no 5pp 172ndash177 2013

[8] L-P Yap H Sancheti M D Ybanez J Garcia E Cadenas andDHan ldquoDetermination ofGSHGSSG andGSNOusingHPLCwith electrochemical detectionrdquo Methods in Enzymology vol473 pp 137ndash147 2010

[9] Z D Zhou and T M Lim ldquoRoles of glutathione (GSH) indopamine (DA) oxidation studied by improved tandem HPLCplus ESI-MSrdquo Neurochemical Research vol 34 no 2 pp 316ndash326 2009

[10] M Yan G-B Shi Y Sui T Guo J-W Zhang and S-J FanldquoDetermination of reduced glutathione in human plasma byRP-HPLCrdquo Pharmaceutical Journal of Chinese Peoplersquos Libera-tion Army vol 3 pp 251ndash253 2008

[11] P Zhu T Oe and I A Blair ldquoDetermination of cellular redoxstatus by stable isotope dilution liquid chromatographymassspectrometry analysis of glutathione and glutathione disulfiderdquoRapid Communications in Mass Spectrometry vol 22 no 4 pp432ndash440 2008

[12] L Bergstrom ldquoSome pathologies of sensory and neural hearinglossrdquo Canadian Journal of Otolaryngology Supplement vol 2pp 1ndash28 1975

[13] L R Snyder J J Kirkland and J L Glajch Practical HPLCMethod Development John Wiley amp Sons Hoboken NJ USA1997

[14] ldquoQ2b validation of analytical procedures methodologyrdquo inProceedings of the International Conference on Harmonization(ICH rsquo97) p 27463 US FDA Federal Register May 1997

[15] G A Shabir ldquoValidation of high-performance liquid chro-matography methods for pharmaceutical analysis understand-ing the differences and similarities between validation require-ments of the US Food and Drug Administration the USPharmacopeia and the International Conference on Harmo-nizationrdquo Journal of Chromatography A vol 987 no 1-2 pp 57ndash66 2003

[16] M Bakshi and S Singh ldquoDevelopment of validated stability-indicating assay methodsmdashcritical reviewrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 28 no 6 pp 1011ndash10402002

[17] A Riboulet-Chavey A Pierron I Durand J Murdaca JGiudicelli and E Van Obberghen ldquoMethylglyoxal impairs theinsulin signaling pathways independently of the formation ofintracellular reactive oxygen speciesrdquoDiabetes vol 55 no 5 pp1289ndash1299 2006

[18] Y-D Hsuuw C-K Chang W-H Chan and J-S Yu ldquoCur-cumin prevents methylglyoxal-induced oxidative stress andapoptosis in mouse embryonic stem cells and blastocystsrdquoJournal of Cellular Physiology vol 205 no 3 pp 379ndash386 2005

RETRACTED

Scientifica 5

Table 4 Summary of robustness studies

Experimental conditions Variation RT (min) ofPeaks 1 2 and 3

Combined peakareas Tailing factor

Change in gradient time(min)

18 622 1022 amp 1325 58992 12020 (nominal) 752 1102 amp 1418 55002 120

22 829 1189 amp 1498 54999 121

Buffer pH

248 751 1112 amp 1421 55022 12225 (nominal) 752 1102 amp 1419 55002 120

252 752 1108 amp 1417 55462 122

Wavelength (nm)282 750 1106 amp 1428 56734 123

280 (nominal) 752 1102 1435 55002 120278 755 1107 1444 53878 120

Flow rate (mLmin)

06 1035 1422 amp 1785 55474 11808 (nominal) 752 1102 amp 1444 55002 120

10 56 862 amp 1128 54878 123

Injection volume (120583L)

48 749 1099 amp 1484 53998 12150 (nominal) 752 1102 amp 1447 55002 120

52 750 1101 amp 1425 56878 121

Column type

Zorbax C8 766 1132 amp 1424 52345 125

Eclipse XDB C8 752 1102 amp 1444 55002 120Zorbax C

18 792 1192 amp 1465 60233 149Note RT retention time Peak 1 is 2-nitro-5-mercapto-benzoic acid Peak 2 is glutathione dimer and Peak 3 is Ellmanrsquos reagent

0

50

100

150

200

250

300

350

400

450

500

Peak

area

(vol

ts)

50 100 150 200 250 300 3500Time (minutes)

GSH (raw)GSH in PC-12 cells

Figure 5 Stability studies of glutathione over a time period of 6 h

325 Biomedical Application Glutathione is required for thedetoxification ofmethylglyoxal a toxicmetabolite of liver It isevident from earlier reports that methylglyoxal accumulatedin cells due to GSH depletion is the major cause for cellulardysfunction and oxidative stress [17] Such stress is alsoobserved in conditions such as inflammation and the cellcopes with the stress with intracellular antioxidants GSH isa major antioxidant present in cells and it exists in reduced

as well as oxidized form depending upon the oxidative stateof the cell An accurate measurement of intracellular GSHprovides a means of determining the oxidative stress causedby an agent and the cellular response to it Bothmethylglyoxal(Figure 3(b)) and CoCl

2(Figure 3(c)) treatment significantly

altered the level of intracellular GSH compared to control(Figure 3(a)) It is possible that CoCl

2also activates GSH

synthesizing enzymes resulting in a higher level of GSHFurther studies will need to be done to test this possibilityThere is a previous report that methylglyoxal interferes withGSH synthesis and secretion [18] which could be whymethylglyoxal treated cells show a lower level of GSH com-pared to control and CoCl

2treated cells The present study

was performed twice with the same sample procedure andHPLC chromatographic conditions When compared withthe control in both the studies authors found a significantdecrease in GSH upon methylglyoxal treatment Furtherexperimentation is needed to study the more accurate andprecise results

4 Conclusion

A simple easy and reliable LC method was developed andvalidated as per the standard guidelines for the determinationof GSH in PC-12 cells A gradient time of 20min with asegmented gradient range and flow rate was found optimumwhen a C

8column was used The validation parameters

showed satisfactory linearity in the range of 1ndash20120583gmL witha high degree of precision and accuracy To the best of ourknowledge this is the first report on measurement of GSH inPC-12 cells upon treatment with these reagents furthermore

RETRACTED

6 Scientifica

the assay leads its applicability to study the role of GSH inpreventing cellular stress

Competing Interests

The authors declare that they have no competing interests

References

[1] N Gadoth and H H Gobel Oxidative Stress and Free RadicalDamage in Neurology Humana Press 2010

[2] B Sharma S Singh and N J Siddiqi ldquoBiomedical implicationsof heavy metals induced imbalances in redox systemsrdquo BioMedResearch International vol 2014 Article ID 640754 26 pages2014

[3] J E Ash S Budavari M OrsquoNeill A Smith P E Heckelmanand J KinnearyTheMerck Index Chapman amp Hall New YorkNY USA 11th edition 1996

[4] A Pompella A Visvikis A Paolicchi V De Tata and A FCasini ldquoThe changing faces of glutathione a cellular protago-nistrdquo Biochemical Pharmacology vol 66 no 8 pp 1499ndash15032003

[5] A Pastore F Piemonte M Locatelli et al ldquoDeterminationof blood total reduced and oxidized glutathione in pediatricsubjectsrdquo Clinical Chemistry vol 47 no 8 pp 1467ndash1469 2001

[6] M C Reed R L Thomas J Pavisic S J James C MUlrich andH FNijhout ldquoAmathematicalmodel of glutathionemetabolismrdquo Theoretical Biology and Medical Modelling vol 5pp 8ndash10 2008

[7] T Santa ldquoRecent advances in analysis of glutathione in bio-logical samples by high-performance liquid chromatography abrief overviewrdquo Drug Discoveries and Therapeutics vol 7 no 5pp 172ndash177 2013

[8] L-P Yap H Sancheti M D Ybanez J Garcia E Cadenas andDHan ldquoDetermination ofGSHGSSG andGSNOusingHPLCwith electrochemical detectionrdquo Methods in Enzymology vol473 pp 137ndash147 2010

[9] Z D Zhou and T M Lim ldquoRoles of glutathione (GSH) indopamine (DA) oxidation studied by improved tandem HPLCplus ESI-MSrdquo Neurochemical Research vol 34 no 2 pp 316ndash326 2009

[10] M Yan G-B Shi Y Sui T Guo J-W Zhang and S-J FanldquoDetermination of reduced glutathione in human plasma byRP-HPLCrdquo Pharmaceutical Journal of Chinese Peoplersquos Libera-tion Army vol 3 pp 251ndash253 2008

[11] P Zhu T Oe and I A Blair ldquoDetermination of cellular redoxstatus by stable isotope dilution liquid chromatographymassspectrometry analysis of glutathione and glutathione disulfiderdquoRapid Communications in Mass Spectrometry vol 22 no 4 pp432ndash440 2008

[12] L Bergstrom ldquoSome pathologies of sensory and neural hearinglossrdquo Canadian Journal of Otolaryngology Supplement vol 2pp 1ndash28 1975

[13] L R Snyder J J Kirkland and J L Glajch Practical HPLCMethod Development John Wiley amp Sons Hoboken NJ USA1997

[14] ldquoQ2b validation of analytical procedures methodologyrdquo inProceedings of the International Conference on Harmonization(ICH rsquo97) p 27463 US FDA Federal Register May 1997

[15] G A Shabir ldquoValidation of high-performance liquid chro-matography methods for pharmaceutical analysis understand-ing the differences and similarities between validation require-ments of the US Food and Drug Administration the USPharmacopeia and the International Conference on Harmo-nizationrdquo Journal of Chromatography A vol 987 no 1-2 pp 57ndash66 2003

[16] M Bakshi and S Singh ldquoDevelopment of validated stability-indicating assay methodsmdashcritical reviewrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 28 no 6 pp 1011ndash10402002

[17] A Riboulet-Chavey A Pierron I Durand J Murdaca JGiudicelli and E Van Obberghen ldquoMethylglyoxal impairs theinsulin signaling pathways independently of the formation ofintracellular reactive oxygen speciesrdquoDiabetes vol 55 no 5 pp1289ndash1299 2006

[18] Y-D Hsuuw C-K Chang W-H Chan and J-S Yu ldquoCur-cumin prevents methylglyoxal-induced oxidative stress andapoptosis in mouse embryonic stem cells and blastocystsrdquoJournal of Cellular Physiology vol 205 no 3 pp 379ndash386 2005

RETRACTED

6 Scientifica

the assay leads its applicability to study the role of GSH inpreventing cellular stress

Competing Interests

The authors declare that they have no competing interests

References

[1] N Gadoth and H H Gobel Oxidative Stress and Free RadicalDamage in Neurology Humana Press 2010

[2] B Sharma S Singh and N J Siddiqi ldquoBiomedical implicationsof heavy metals induced imbalances in redox systemsrdquo BioMedResearch International vol 2014 Article ID 640754 26 pages2014

[3] J E Ash S Budavari M OrsquoNeill A Smith P E Heckelmanand J KinnearyTheMerck Index Chapman amp Hall New YorkNY USA 11th edition 1996

[4] A Pompella A Visvikis A Paolicchi V De Tata and A FCasini ldquoThe changing faces of glutathione a cellular protago-nistrdquo Biochemical Pharmacology vol 66 no 8 pp 1499ndash15032003

[5] A Pastore F Piemonte M Locatelli et al ldquoDeterminationof blood total reduced and oxidized glutathione in pediatricsubjectsrdquo Clinical Chemistry vol 47 no 8 pp 1467ndash1469 2001

[6] M C Reed R L Thomas J Pavisic S J James C MUlrich andH FNijhout ldquoAmathematicalmodel of glutathionemetabolismrdquo Theoretical Biology and Medical Modelling vol 5pp 8ndash10 2008

[7] T Santa ldquoRecent advances in analysis of glutathione in bio-logical samples by high-performance liquid chromatography abrief overviewrdquo Drug Discoveries and Therapeutics vol 7 no 5pp 172ndash177 2013

[8] L-P Yap H Sancheti M D Ybanez J Garcia E Cadenas andDHan ldquoDetermination ofGSHGSSG andGSNOusingHPLCwith electrochemical detectionrdquo Methods in Enzymology vol473 pp 137ndash147 2010

[9] Z D Zhou and T M Lim ldquoRoles of glutathione (GSH) indopamine (DA) oxidation studied by improved tandem HPLCplus ESI-MSrdquo Neurochemical Research vol 34 no 2 pp 316ndash326 2009

[10] M Yan G-B Shi Y Sui T Guo J-W Zhang and S-J FanldquoDetermination of reduced glutathione in human plasma byRP-HPLCrdquo Pharmaceutical Journal of Chinese Peoplersquos Libera-tion Army vol 3 pp 251ndash253 2008

[11] P Zhu T Oe and I A Blair ldquoDetermination of cellular redoxstatus by stable isotope dilution liquid chromatographymassspectrometry analysis of glutathione and glutathione disulfiderdquoRapid Communications in Mass Spectrometry vol 22 no 4 pp432ndash440 2008

[12] L Bergstrom ldquoSome pathologies of sensory and neural hearinglossrdquo Canadian Journal of Otolaryngology Supplement vol 2pp 1ndash28 1975

[13] L R Snyder J J Kirkland and J L Glajch Practical HPLCMethod Development John Wiley amp Sons Hoboken NJ USA1997

[14] ldquoQ2b validation of analytical procedures methodologyrdquo inProceedings of the International Conference on Harmonization(ICH rsquo97) p 27463 US FDA Federal Register May 1997

[15] G A Shabir ldquoValidation of high-performance liquid chro-matography methods for pharmaceutical analysis understand-ing the differences and similarities between validation require-ments of the US Food and Drug Administration the USPharmacopeia and the International Conference on Harmo-nizationrdquo Journal of Chromatography A vol 987 no 1-2 pp 57ndash66 2003

[16] M Bakshi and S Singh ldquoDevelopment of validated stability-indicating assay methodsmdashcritical reviewrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 28 no 6 pp 1011ndash10402002

[17] A Riboulet-Chavey A Pierron I Durand J Murdaca JGiudicelli and E Van Obberghen ldquoMethylglyoxal impairs theinsulin signaling pathways independently of the formation ofintracellular reactive oxygen speciesrdquoDiabetes vol 55 no 5 pp1289ndash1299 2006

[18] Y-D Hsuuw C-K Chang W-H Chan and J-S Yu ldquoCur-cumin prevents methylglyoxal-induced oxidative stress andapoptosis in mouse embryonic stem cells and blastocystsrdquoJournal of Cellular Physiology vol 205 no 3 pp 379ndash386 2005