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TRANSCRIPT
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
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
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