Speciation and Quantitation of Underivatized and Ellman's Derivatized Biological Thiols and...

ANALYTICAL BIOCHEMISTRY 242, 136–144 (1996)ARTICLE NO. 0439

Speciation and Quantitation of Underivatized and Ellman’sDerivatized Biological Thiols and Disulfidesby Capillary Electrophoresis

James Russell and Dallas L. Rabenstein1

Department of Chemistry, University of California—Riverside, Riverside, California 92521

Received March 13, 1996

been correlated directly with the progression towardCapillary electrophoresis (CE) methods have been de- chronic heart disease (7, 8). Cystine is a nutrient which

veloped for the speciation and quantitation of thiols and the erythrocyte transports and reduces to cysteine,disulfides of biological interest, including the endoge- which acts as a substrate for g-glutamyl-cysteinyl syn-nous compounds glutathione, glutathione disulfide, cys- thetase (9). In addition to endogenous thiols and disul-teine, cystine, homocysteine, and homocystine and the fides, there are exogenously added thiol-containingtherapeutic agents penicillamine, penicillamine disul- therapeutic agents such as captopril, penicillamine,fide, N-acetylcysteine, and captopril. Good speciation and N-acetylcysteine; metabolic products of these ther-and quantitation were achieved for the underivatized apeutic agents include disulfides. Thus, it is of interestthiols and disulfides using a detection wavelength of 200 to speciate and quantitate both the thiol and disulfidenm; detection limits were in the range 20–90 mM (1–4 forms of thiol-containing endogenous compounds andpmol) using a 50-mm-diameter capillary. To achieve therapeutic agents.lower detection limits, thiols were derivatized with the

The literature shows that the analysis of thiols andthiol-specific probe molecule, 5,5*-dithio-bis-(2-nitroben-disulfides has been achieved by several techniques,zoic acid) (Ellman’s reagent). Good speciation and quan-principally by HPLC using uv/visible (10–18), fluores-titation were achieved for the Ellman’s derivatized thi-cence (19–25), or electrochemical detection (26–31).ols using a detection wavelength of 357 nm; detectionThiol/disulfide content has also been measured by en-limits were in the range 5–50 mM (0.03–0.3 pmol) usingzyme assays (32–36) and Raman spectroscopy (37, 38).a 25-mm-diameter capillary. Both the underivatized andAnalysis of thiols and disulfides using capillary electro-derivatized methods were applied to the determinationphoresis (CE)2 has been limited to endogenous com-of glutathione in human erythrocytes. Glutathione con-pounds (39) or to the thiol forms (40, 41). Comparedcentrations of 2–3 mM were obtained for the erythrocyte

samples analyzed, with good agreement between results to HPLC and other forms of analysis, CE offers theobtained by the two methods. q 1996 Academic Press, Inc. advantages of excellent resolving power, fast analysis,

and ease of automation and it is not sample or solventintensive (42).

In this paper, we report CE methods with which indi-vidual compounds in mixtures of biologically and phar-Thiol-containing compounds and their corresponding

disulfides are of importance in a variety of diseases macologically important thiol-containing compoundsand their disulfides can be determined. The primarywhich possess an oxidative etiology, such as chronic

heart disease (1, 2), rheumatoid arthritis (3), and more aim of this research was to achieve speciation andquantitation of underivatized thiols and disulfides byrecently autoimmune deficiency syndrome (AIDS) (4,

5). In vivo, there exists a complicated mixture of thiols CE with uv detection. However, intrinsic to the deter-mination of underivatized thiols and disulfides is anand disulfides, each of which has its own particular

relevance. The ratio of oxidized to reduced glutathione inherent lack of sensitivity, due to their uv absorbancehas been shown to be an effective measure of the oxida-tive stress on a system (6). Homocysteine levels have

2 Abbreviations used: CE, capillary electrophoresis; EOF, elec-troosmotic flow; GSH, glutathione; HPLC, high performance liquidchromatography; ESSE, Ellman’s reagent.1 To whom correspondence should be addressed. Fax: (909) 787-4713.

136 0003-2697/96 $18.00Copyright q 1996 by Academic Press, Inc.

All rights of reproduction in any form reserved.

AID AB 9776 / 6m20$$$$41 10-04-96 09:34:19 aba AP: Anal Bio

ELECTROPHORETIC DETERMINATION OF THIOLS AND DISULFIDES 137

FIG. 1. Capillary electropherogram of a mixture of endogenous thiols and disulfides. The concentrations were homocystine, 0.175 mM;homocysteine, 0.85 mM; cystine, 0.75 mM; cysteine, 1.5 mM; glutathione disulfide, 0.125 mM; and glutathione, 0.175 mM. The CE conditionswere 0.1 M phosphate buffer (pH 2.3) and positive to negative polarity at 15 kV. The absorbance was measured at 200 nm.

characteristics. This problem was alleviated for the able-wavelength detection was used. Various fused sil-thiol forms of the compounds studied by derivatization ica capillary cartridges of different dimensions with awith Ellman’s reagent (5,5*-dithio-bis-2-nitrobenzoic polyacrylamide coating were used. A 50 cm 1 50-mm-acid), a thiol-specific probe molecule which contains a i.d. capillary was used for separation of underivatizedchromophore (43). Ellman’s reagent reacts with thiols thiols and disulfides, and a 24 cm1 25-mm-i.d. capillaryto form uv-absorbing mixed disulfides which allow de- was used for thiols derivatized with Ellman’s reagent.tection at a wavelength of 357 nm (44–47). Capillary The analysis of erythrocyte glutathione by the underi-electropherograms measured for a sample by both the vatized method was achieved using a 50 cm 1 50-mmunderivatized and derivatized methods provide specia- user-assembled, fused bare silica capillary (Polymicro-tion information about the thiols and disulfides in the technologies Inc., Phoenix, AZ). Samples were intro-mixture. duced onto the capillaries by pressure injection (5 psi/

s for 3 s). The sample injection volumes were 6.3 andMATERIALS AND METHODS 45 nl for the 25- and 50-mm-diameter capillaries, re-

spectively. The detection of underivatized species wasMaterials. Sodium phosphate was purchased fromachieved by measuring the absorbance at 200 nm. TheFisher Scientific (New Jersey). Glutathione, glutathi-Ellman’s derivatized compounds were detected by mon-one disulfide, cysteine, cystine, homocysteine, homocys-itoring at 200, 325, and 357 nm. Peak heights and peaktine, D-penicillamine, D-penicillamine disulfide, N-areas were determined with the Bio-Rad software.acetylcysteine, 2-mercaptoacetic acid, 2-mercapto-

propionic acid, and dithiothreitol were obtained from Capillary electrophoresis of underivatized thiols andSigma Chemical Co. Captopril was obtained from E. R. disulfides. Homocysteine, homocystine, cysteine, cys-Squibb & Sons Inc. 5,5*-Dithio-bis-(2-nitrobenzoic acid) tine, D-penicillamine, D-penicillamine disulfide, gluta-was obtained from Eastman Kodak Co. thione, and glutathione disulfide were prepared as 1

mM solutions in the run buffer used in the CE separa-Equipment. A Biofocus 3000 capillary electrophore-sis system (Bio-Rad, Hercules, CA) with fast-scan, vari- tion. Capillary electropherograms were measured for


RUSSELL AND RABENSTEIN138

TABLE 1

Migration Times, Precision of Peak Areas, Detector Calibration Data,and Detection Limits for Underivatized Thiols and Disulfides

Relative standard DetectionMigration deviation of peak area Correlation limit

Compounda timeb (min) (%)c Sloped Interceptd coefficient (mM)e

Homocystine 4.13 { 0.09 5.90 2.4 0.00012 0.9988 46Homocysteine 4.76 { 0.09 6.60 1.6 0.00016 0.9982 44N-Acetyl-cysteine f 9.98 { 0.31 11.0 5.4 00.00012 0.9957 65Dithiothreitol f 10.23 { 0.18 5.24 5.6 0.00014 0.9994 28Captopril f 11.17 { 0.39 2.57 1.5 0.00012 0.9982 90Cystine 17.50 { 0.31 3.58 2.1 0.00009 0.9975 66Cysteine 18.49 { 0.41 2.54 1.6 0.00013 0.9937 69D-Penicillamine 19.25 { 0.14 1.99 2.5 0.00009 0.9993 86D-Penicillamine disulfide 20.33 { 0.18 6.37 11.1 0.00013 0.9991 29Glutathione 24.45 { 0.45 5.53 10.9 0.00001 0.9983 25Glutathione disulfide 22.39 { 0.17 3.57 23.2 0.00003 0.9926 20

a Unless specified otherwise, a 15-kV positive to negative polarity was used with a 0.1 M phosphate run buffer (pH 2.3).b Each result is the average of eight measurements; uncertainties are standard deviations.c Relative standard deviation of eight measurements.d y Å (slope)x / intercept, where y is the peak height (in absorbance units at 200 nm) and x is the concentration (in mol/liter). The slope

has units of absorbance units liters/mol.e The criteria used to establish the detection limits are given in the text.f A 19-kV negative to positive polarity was used with a 0.01 M phosphate run buffer (pH 2.3).

these solutions on a 50 cm 1 50-mm-i.d. capillary by min until equilibrium was reached (25, 29). Capillaryelectropherograms were measured for each derivatizedpressure injection (5 psi) and application of a 15-kV

potential, using positive to negative polarity and a run compound on a 24 cm 1 25-mm-i.d. coated capillaryusing pressure injection, followed by application of abuffer of 0.1 M phosphate (pH 2.3), which was prepared

by adjusting the pH of a 0.1 M solution of NaH2PO4 to 12-kV potential, using negative to positive polarity. Ho-mocysteine was derivatized in a similar manner, andpH 2.3 with HCl. A minimum of eight replicate elec-

tropherograms were measured for each compound to capillary electropherograms were measured using a 19-kV positive to negative polarity. To determine the stan-determine the standard deviation of migration times

and peak areas. dard deviation of the migration time and the relativestandard deviation of the peak area, eight replicateN-Acetylcysteine, dithiothreitol, and captopril were

prepared as 1 mM solutions in a 0.01 M phosphate run electropherograms were recorded for each compound.To calibrate detector response, capillary electrophero-buffer (pH 2.3). These compounds were injected as de-

scribed above onto the 50 cm 1 50-mm-i.d. capillary grams were measured for each compound at thiol con-centrations ranging from 1 mM to 1 mM.and were separated by application of a 19-kV potential,

using a negative to positive polarity and a run buffer Analysis of erythrocyte glutathione by the underiva-of 0.01 M phosphate (pH 2.3), which was prepared by tized method. Fresh blood samples were collecteddilution of the 0.1 M phosphate run buffer. Eight repli- from healthy volunteers in anticoagulant lithium–hep-cate electropherograms were measured for each com- arin vacutainers. Blood samples were centrifuged atpound. 2500 rpm for 5 min and the plasma layer was removed.

To calibrate detector response, capillary electropher- The packed erythrocytes were then washed twice withograms were measured for each compound at concen- equal volumes of isotonic saline solution. The erythro-trations ranging from 1 mM to 5 mM. cytes were lysed by freezing in liquid nitrogen followed

by thawing in a water bath at 607C (40). This procedureCapillary electrophoresis of thiols after derivatizationwith Ellman’s reagent. Captopril, glutathione, cys- was repeated three times to ensure complete cell lysis.

One milliliter of the lysed erythrocytes was added to 1teine, and D-penicillamine were prepared as 1 mM solu-tions in 0.01 M sodium phosphate buffer (pH 7.4). Ell- ml of chilled 0.1 M trichloroacetic acid solution con-

taining 1.0 M EDTA. The sample was vortex mixed forman’s reagent was prepared as a 3 mM solution in 0.01M sodium phosphate buffer (pH 7.4). One hundred mi- 5 min and then centrifuged at 3500 rpm for 5 min to

remove protein precipitate. The supernatant was with-croliters of the thiol-containing solution was added to100 ml of Ellman’s reagent in an Eppendorf tube. The drawn and filtered through a 0.2-mm filter. Twenty mi-

croliters of the filtrate was added to 80 ml of the phos-reaction mixture was left at room temperature for 20



TABLE 2

Migration Times, Precision of Peak Areas, Detector Calibration Data, and Detection Limits for Derivatized Thiols

Relative standard DetectionMigration deviation of peak area Correlation limit

Compounda timeb (min) (%)c Sloped Interceptd coefficient (mM)e

Ellman’s anion 1.58 { 0.06 9.18 6.8 00.000080 0.9986 48Ellman’s reagent 2.26 { 0.05 4.29 7.4 0.000104 0.9987 14Captopril 2.91 { 0.12 5.84 3.2 0.000115 0.9956 16Glutathione 3.06 { 0.02 9.75 7.1 0.000348 0.9976 5Cysteine 3.42 { 0.18 4.78 3.3 0.000036 0.9996 28D-Penicillamine 3.68 { 0.24 3.67 1.3 0.000030 0.9864 59Homocysteine f 4.49 { 0.17 5.13 7.4 0.000062 0.9942 12Mercaptoacetic acid 1.95 { 0.06 6.97Mercaptopropionic acid 2.13 { 0.07 6.97

a Unless specified otherwise, a 12-kV negative to positive polarity was used with a 0.01 M phosphate run buffer (pH 7.4).b Uncertainties are standard deviations.c Relative standard deviation of eight measurements.d y Å (slope)x / intercept, where y is the peak height (in absorbance units at 357 nm) and x is the concentration (in mol/liter). The slope

has units of absorbance units liters/mol.e The criteria used to establish the detection limits are given in the text.f A 19-kV positive to negative polarity was used with the same run buffer.

phate run buffer (pH 2.3), to provide a particle-free cedure was similar to that described in the previoussection. Recoveries of 99 { 2% and 96 { 2% were ob-sample solution for injection onto the CE system.

Recovery studies were done by spiking erythrocyte tained for the two erythrocyte samples.To compare the results obtained by the underivatizedsamples from two donors with exogenous GSH. The

procedure involved addition of 50 ml of a 0.010 M GSH and derivatized methods, glutathione was determinedin erythrocytes from six donors. Erythrocytes from twosolution to 1.00 ml of packed erythrocytes to give an

added GSH concentration of 0.48 mM. The spiked of the donors were each analyzed in parallel by the twomethods. The samples from the four other donors wereerythrocytes were then lysed, the protein was precipi-

tated and the sample was filtered, as described above. analyzed first by the underivatized method. The follow-ing week, new samples were obtained from the sameThe supernatant was then analyzed for GSH. The

spiked samples were analyzed in parallel with un- four donors and analyzed by the Ellman’s derivatizedmethod.spiked samples, and the percent recovery was deter-

mined from the difference in concentrations found forthe spiked and unspiked samples. Recovery studies

RESULTS AND DISCUSSIONwere done in duplicate for erythrocytes from each do-nor; recoveries of 101 { 3% and 96 { 4% were obtained Analysis of thiols and disulfides by the underivatizedfor the two erythrocyte samples. method. A 50-cm capillary was chosen for separation

of underivatized thiols and disulfides because of theAnalysis of erythrocyte glutathione by the Ellman’sderivatized method. Erythrocytes were separated greater selectivity of longer capillaries (48). The 50 cm

1 50-mm-i.d. capillary was found to have optimal di-from plasma and washed with isotonic saline solutionas described above. One milliliter of packed erythro- mensions for the selectivity and sensitivity required for

the various separations. A low pH (2.3) run buffer wascytes was added to 1 ml of saline containing 1.0 M

EDTA. The erythrocytes were then lysed by repeatedly used for two reasons. First, thiol/disulfide exchange isquenched at low pH due to protonation of the thiolatefreezing and thawing the sample as described above.

The resulting suspension was centrifuged at 3500 rpm species (49). Thus, thiol and disulfide concentrationsin thiol/disulfide mixtures will be stable as a functionfor 5 min to precipitate the protein and membrane frac-

tions. The supernatant was removed and filtered of time. Second, migration times are longer and selec-tivity is increased at lower pH due to decreased elec-through a 0.2-mm filter. Twenty microliters of the re-

sulting particle-free solution was added to 80 ml of a 5 troosmotic flow (EOF) (48).Most of the thiols and disulfides to be separated aremM solution of Ellman’s reagent. After 20 min of reac-

tion, the sample was analyzed by CE. cations at pH 2.3 and thus a positive to negative polar-ity was applied. A run buffer concentration of 0.1 MRecovery studies were done by spiking packed eryth-

rocytes from two donors with exogenous GSH; the pro- phosphate and a potential of 15 kV were found to be



optimum parameters for the separation. Those com-pounds which were separated using a negative to posi-tive polarity are either weakly charged or neutral atpH 2.3, and thus a lower ionic strength was used to-gether with a higher potential of 19 kV to move thecompounds through the capillary without losing sensi-tivity or selectivity.

An electropherogram showing the separation of amixture of endogenous thiols and disulfides is shownin Fig. 1. The concentrations of the various compoundswere in the 0.125 to 1.5 mM range. To determine thereproducibility of migration times and peak areas,eight replicate electropherograms were measured foreach compound separately. Migration times and theirstandard deviations are reported in Table 1. Also re-ported in Table 1 are relative standard deviations forthe peak areas.

To determine detection limits and to calibrate detec-tor response, electropherograms were measured foreach compound over the concentration range 1 mM–5mM. Detection limits are reported in Table 2; the detec-tion limit is taken to be the concentration at a signal-to-noise ratio of 3:1, where the noise is peak-to-peaknoise. Plots of peak area and peak height vs concentra-tion were linear over the concentration range from thedetection limit to 5 mM. The data were fit by linearleast-squares methods; the calibration parameters ob-tained using the peak height data together with corre-lation coefficients are reported in Table 1.

Analysis of thiols by the Ellman’s derivatized method.Detection limits for the underivatized method are rela-tively high due to the absorbance characteristics of thecompounds. To obtain lower detection limits for thethiols, they were derivatized with Ellman’s reagent,a thiol-specific reagent which contains a uv-absorbingchromophore which can be detected at 357 nm, wherethe noise level is lower. Ellman’s reagent has been usedextensively to study and quantitate thiol-containingcompounds in biological systems (43–47).

FIG. 2. Capillary electropherograms of the products of the reactionThe stepwise reactions of Ellman’s reagent (ESSE) between Ellman’s reagent and glutathione at various ratios withwith the thiol RSH are given in Eqs. [1] and [2] (43). detection at 357 nm (a–e) and 200 nm (f–j). (a, f) 2 mM ESSE:0.2

mM GSH; (b, g) 1 mM ESSE:0.2 mM GSH; (c, h) 0.4 mM ESSE:0.2ESSE reacts with RSH to form Ellman’s anion (ES0)mM GSH; (d, i) 0.2 mM ESSE:0.2 mM GSH; (e, j) 0.1 mM ESSE:0.2and the mixed disulfide ESSR, which in turn reactsmM GSH. The CE conditions were 0.01 M phosphate buffer (pH 7.4)with another molecule of RSH to form ES0 and RSSR.and negative to positive polarity at 12 kV.

ESSE / RSH S ESSR / ES0 / H/ [1]

ESSR / RSH S RSSR / ES0 / H/ [2] on a coated 24 cm 1 25-mm-i.d. capillary, which pro-vided a fast and efficient separation of the various spe-cies. The anionic nature of the mixed disulfides dictatedWhen Ellman’s reagent is in excess, the reaction

reaches equilibrium at Eq. [1] and the major products that the separation be achieved using a low-ionic-strength buffer at a potential of 12 kV and negativeof the reaction are ES0, which has a lmax at 412 nm,

ESSR, which has a lmax at 357 nm, and excess Ellman’s to positive polarity. The homocyteine–Ellman’s mixeddisulfide was separated at 19 kV using positive to nega-reagent, which has lmax at 325 nm (46). These absorp-

tion bands are broad and overlap. tive polarity. Because the products of the reaction ofEllman’s reagent with thiols are sensitive to decompo-ESSE, ES0, and the mixed disulfides were separated



mM Ellman’s reagent for 20 min, and then the reactionmixtures were separated by CE with detection at 200and 357 nm. The results are presented in Figs. 2 and3. The electropherograms measured at 200 nm in Fig.2 show that, at low ratios of ESSE to GSH, the excessGSH reacts with ESSG by Eq. [2] to form oxidized glu-tathione (GSSG). The results in Fig. 3 indicate com-plete reaction of glutathione with ESSE at anESSE:glutathione ratio of 5:1.

The electropherogram in Fig. 4 shows the separationachieved for a mixture of thiols which was derivatized byreaction with ESSE. Migration times and their standarddeviations were determined by measuring eight replicatesfor each compound separately (see Table 2). Also reportedare relative standard deviations for the peak areas.

Electropherograms were measured for the variouscompounds over the concentration range 1 mM–1 mM

FIG. 3. Normalized peak height for the Ellman’s reagent–glutathi- to determine detection limits and to calibrate detectorone mixed disulfide (ESSG) as a function of the Ellman’s reagent:glu- response. Detection limits are reported in Table 2; detec-tathione ratio. Data are from capillary electropherograms a–e in

tion limits were typically lower for the Ellman’s deriva-Fig. 2.tized thiols than the underivatized thiols and disulfidesdue to the absorption characteristics of the derivatizedthiols and the lower noise in capillary electrophero-sition at high pH (45) and are insoluble at pH less thangrams measured with detection at 357 nm. Calibration7.4, a pH 7.4 sodium phosphate run buffer was used.plots of peak area and peak height vs concentration wereTo establish appropriate reaction conditions for deriv-linear over the concentration range from the detectionatization of thiols with Ellman’s reagent, 0.2 mM gluta-

thione (GSH) was reacted with 0.1, 0.2, 0.4, 1.0, and 2.0 limit up to 1 mM, except for mercaptoacetic acid and

FIG. 4. Capillary electropherogram of a mixture of Ellman’s derivatized thiols. The concentrations were mercaptoacetic acid, 1 mM;mercaptopropionic acid, 1 mM; captopril, 0.05 mM; glutathione, 0.01 mM; cysteine, 0.05 mM; D-penicillamine, 0.25 mM; Ellman’s reagent, 4mM; and homocysteine, 0.3 mM. The CE conditions were 0.01M sodium phosphate buffer (pH 7.4) and negative to positive polarity at 12kV, and the absorbance was measured at 357 nm. Conditions for the inset capillary electropherogram were 0.01 M sodium phosphate buffer(pH 7.4) and positive to negative polarity at 19 kV, and the absorbance was measured at 357 nm.



TABLE 3 shown in Fig. 5. The results obtained from six donorsare reported in Table 4. A good correlation was foundElectrophoretic Mobilities of Underivatizedbetween the two methods. The recoveries obtained byand Derivatized Compoundsanalysis of erythrocytes which were spiked with a

meffectivea,b meffective

a,bknown concentration of glutathione prior to lysis by

Compound (underivatized) (derivatized) both the underivatized and derivatized methods indi-cate that there is not a significant loss of glutathioneHomocysteine 3.476 1 1002 4.249 1 1003

Homocystine 2.995 1 1002 during sample workup or in the capillary. The erythro-Cystine 6.870 1 1003 cyte glutathione concentrations in Table 4 of betweenCysteine 6.555 1 1003 01.004 1 1002

2 and 3 mM are in agreement with published valuesD-Penicillamine 6.235 1 1003 09.675 1 1003

determined using other techniques (11, 51–55). In con-D-Penicillamine disulfide 5.821 1 1003

trast, a previous attempt to determine erythrocyte glu-Glutathione disulfide 5.142 1 1003

Glutathione 4.578 1 1003 01.158 1 1002 tathione by CE reported values in the range of 1.2 mMAcetyl cysteine 02.927 1 1003 (40). It is not clear why the results obtained in theDithiothreitol 02.634 1 1003

previous study are lower than our results and litera-Captopril 01.652 1 1003 01.226 1 1002

ture values.Ellman’s anion 02.346 1 1002

Mercaptoacetic acid 01.880 1 1002

Mercaptopropionic acid 01.712 1 1002

SUMMARYEllman’s reagent 01.607 1 1002

Methods for the determination of biological thiolsa Units are V01 cm2 min01.and their disulfides by CE are reported. Both thiolsb meffective is negative for species that are fractionally present as

anions.

mercaptopropionic acid, which did not react completelywith ESSE, apparently due to a less favorable equilib-rium. Calibration parameters obtained by fitting thepeak height vs concentration data by linear least-squares methods are reported in Table 2.

Electrophoretic mobilities. To determine the effectof derivatization on the electrophoretic mobilities, theEOF was calculated for each set of conditions using a0.5% mesityl oxide solution as a neutral marker. Theeffective mobilities of the underivatized and deriva-tized species (Table 3) were determined using the rela-tionship that the apparent electrophoretic mobility (ma)is the sum of the EOF (meof) and the effective mobility(me) (50).

ma Å me / meof [3]

The effective mobilities of glutathione, cysteine, peni-cillamine, and captopril are all greater in the deriva-tized form, leading to a faster separation in the Ell-man’s derivatization method. Only homocysteinedisplayed a lower electrophoretic mobility in the deriv-atized form.

Determination of erythrocyte glutathione by CE. To FIG. 5. Capillary electropherograms from the determination oferythrocyte glutathione by the underivatized and Ellman’s deriva-demonstrate application of the CE methods for deter-tized methods. The CE conditions were (upper trace) 0.1 M sodiummination of a thiol in a biological matrix, the concentra-phosphate buffer (pH 2.3) and positive to negative polarity at 15 kV,tion of glutathione in erythrocytes was determined by and the absorbance was measured at 200 nm; (lower trace) 0.01 M

both the derivatized and underivatized methods. Rep- sodium phosphate (pH 7.4) and negative to positive polarity at 12kV, and the absorbance was measured at 357 nm.resentative electropherograms from both analyses are



TABLE 4

Erythrocyte Glutathione Concentrations (mM) Determined by CE

Underivatized Ellman’s derivatized Ellman’s derivatizedthiol measured at thiol measured at thiol measured at

Donor a 200 nmb 325 nmb 357 nmb

1 2.6 { 0.1 2.6 { 0.12 2.3 { 0.1 2.3 { 0.13 2.5 { 0.1 2.4 { 0.2 2.3 { 0.24 2.9 { 0.3 3.2 { 0.2 3.1 { 0.45 2.4 { 0.1 2.0 { 0.1 2.1 { 0.16 2.7 { 0.1 2.7 { 0.2 2.7 { 0.2

a The samples from donors 1 and 2 were each analyzed in parallel by the two methods. The samples from donors 3–6 were each analyzedfirst by the derivatized method. The following week, samples were obtained from the same donors and analyzed by the underivatizedmethod.

b Each result reported for the underivatized method is the average of 4–6 determinations while for the Ellman’s derivatized method eachresult is the average of 10 determinations.

and disulfides can be determined by the underivatized of the glutathione determination by the Ellman’s deriv-atized method (Ç3 min) and the ease of automation ofprocedure; however, interferences from other compo-

nents of a biological matrix are expected to be more of the CE system make this technique attractive for rou-tine clinical determinations of erythrocyte glutathione.a problem with detection at 200 nm. The method in

which the thiols are derivatized by reaction with Ell- The sensitivity of the derivatized CE method with a50-mm-i.d. capillary is also in the concentration rangeman’s reagent has lower detection limits, due to the

absorption properties of the derivative and the lower at which several other endogenous thiols and thiol-con-taining drug molecules are present in plasma and urinenoise in capillary electropherograms measured at 357

nm. Analysis times are also considerably shorter with (24, 25, 56). The sensitivity of the CE methods alsomakes them attractive as analysis methods for thethe derivatization method. The low detection limits in

Tables 1 and 2 combined with the small sample vol- study of the chemistry of biological thiols and disul-fides, e.g., for measurement of the kinetics and equilib-umes injected onto the capillaries allow detection of

minimum quantities in the 1- to 4-pmol range by the ria of thiol/disulfide exchange reactions and for mea-surement of the redox potentials of biological thiols.underivatized method and in the 0.03- to 0.3-pmol

range by the derivatized method. It is important tonote that the detection limits and minimum detectable ACKNOWLEDGMENTSquantities for the derivatized method were obtained

This research was supported by NIH Grant GM-37000 to D.L.R.using a 25-mm-diameter capillary; detection limits low-The authors thank Jacques Prudhomme for taking blood samples.ered by a factor of Ç10 are expected with a 50-mm-i.d.

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