Determination of bilirubin by adsorptive stripping voltammetry

61

J Elecrroanai Chetn, 185 (1985) 61-71 Elsevter Sequoia S A.

Lausanne

DETERMINATION VOETAlblMETRY

J WANG

*, D B LUO

- Pnnted

rn The Netherlands

OF BlLIRUBIN

**

and PA M

BY ADSORPTIVE

FARIAS

l

**

Deporcmenr of Chemrsny, New Mexrco Srare Unroersrry. La

(-

:wed 30th May 1984.

tn rewed

STRIPPING

Crucer, NM 88003

(LJ S A )

form 10th August 1984)

ABSTRACTA stnppmg method for the determtnatton of blllrubm at the subrmcromolar and nanc;molar concentra~lon levels IS described The method IS based on controlled adsorpttqe accumulation of blhrubm at the stattc mercury drop electrode followed by dtfferenuai pulse measurement of the surface spectes After 15 mm preconcentration. a detection hrmt near 5 X lo-” AI bthrubm IS obtaned TJe adzorpuve s~~pplng response IS evaluated wth respect to concentration dependence, preconcentratlon time and potenual, pH, Ionic wenglh the presence of surfactants and other vstlables Best results are obtamed usmg sodmm acetate (pH 8 2) solution A signal enhancement factor of 11 IS observed usmg 5 mm preconcentratlon (compared to the response of s&&on-phase voltammetry) The relatlxe standard dewauon at the 2 x lo-’ M level IS 2 7%

Bkubin (I) is a tetrapyrrole byproduct of hemoglobm rnc tabohsm whose accurate deterrmaatlon rn body flulds IS Important for dlagnostlc purposes and for

therapeutic momtonng Free newborn Infants. Because of system, It has been the focus laboratones measure btibm

blhrubm is a h@ly toxic compound, especially for the great importance of brlu-ubm m the physiologgcal of a large number of chnical mvestigatlons. Chmcal spectrophotometncally, either chrectly OF after a dlazo

* To whom correspondence should be addressed ** UNESCO Scholar Present address Department of Chemtstry, South-Central tnsutute for Nattond Mtnontres, Wuhan. People’s Repubhc of Chma *** Permanent address Department of Chemtstry, Ponttficta Umverstdade Catohca do RIO de Janetro. RIO de Jartetro, Braztl 0022-0728/8S/SO3

30

0 1985 Elsevter Sequota S A

62

reaction

[I]

eiectroanalytrcal procedures have been suggested for the based on Its oxrdation at platrnum or glassy carbon electrodes m drmethylformamtde [2], or Its reductton at the mercury electrode in drmethylsulfoxrde [3] or m aqueous media [4-71 The use of advanced voltammetnc techmques such as differential pulse or square wave polarography has resulted in convetuent quantttatlon at the mlcromolar concentration level [5-71 The reductron of brhrubm utthzed m these measurements, most lrkely involves reduction of the determination

N-H

Varrous

of blhrubm,

bonds on the inner pyrrole nngs [S].

The present work reports a new stnppmg procedure for trace measurement of brhrubm based on Its adsorpttve accumulation at the static mercury drop electrode. Adsorptive stnppmg analysis has been shown to be an important method m trace analysts because of tts broad scope of apphcattons and relatrvely simple mstrumentatlon [g--19] The method consrsts of an mittal preconcentratton of the analyte by controlled adsorption onto the surface of the working eicctrode followed by quantttatron of the accumulated specres by a conventtonal pulse voltammetnc procedure (e g . drfferentral pulse or square wave). Unhke conventronal stnppmg voltammetry, no charge-transfer reaction 1s mvolved in the accumulatton Increased sensrtrvlty (compared to drrect potential pulse measurements) IS obtamed as a result of the prcconcentratton step, and a high degree of selectrvtty IS achteved by transfemng the electrode mto a blank solutron between the preconcentratron and measurement

steps The method can be applied to any electroacke compound that adsorbs strongly, tn a reproductble way, on electrode surfaces. The method has been successfully applied to trace measurements of compounds such as chlorpromazme [8,9], heme [lo], doparrune [ll], codeme and percame 1121, butylated hydroxyamsole [13], 9,1U-phenanthrenequmone [14], adnamycm [15], drazepam and mtrazepam [16] or ctmetrdtne 1171. Adsorption of varrous heavy metal complexes has been exploited for trace measurements of metals, such as cobalt or mckel, which do not form amalgams stgrufrcantly [18,19] Most apphcatrons have used the hanging mercury drop electrode (for reducible species) and carbon or platinum dtsk electrodes (for oxtdrzable specres) The charactenstrcs and advantages of adsorptrve stnppmg analysts of brluubtn are elucrdated m thts paper. EXPERIMENTAL Sohtrons

The

and

PAR

rnstt-unlentatlon

Model

303A static

mercury

drop

electrode

was employed

with an

Ag,/AgCl (saturated KClj reference electrode and a platinum wire auxiliary electrode, and was interfaced to the PAR 264 Analyzer, as recommended by the manufacturer Instrument settings were as follows medium drop srze, equrhbnum time 15 s, potentral scan rate 5 mV/s, pulse amplitude 50 mV, pulse repetrtron, 0.5 s. A PAR X-Y recorder (Model 0073) was used for the collection of experimental data The sample cell wzs PAR Model 0057. Because bllu-ubm IS photosensitrve, the cell was covered WILL alummum fork A magnetic st.rrrer (Troemner Model 500) and a sttrnng bar provrded the convectrve transport dunng the preconcentration.

63

Stock solutrons (1 X lo-’ M) of brlu-ubm (Sigma) were prepared daily by drssolvmg 5 8 mg of bihrubm u-r 5 ml 0.1 A4 sodmm hydroxrde, and a drlutmg to 100 ml wrth 0 1 M sodnrm acetate. The solutrons were stored m the dark at 4°C. All solutions were prepared from deionized water and analyttcal grade reagents. Procedure

A 10 ml volume rf the supportmg electrolyte solutron (usually 0 1 M sodmm acetate) was added to the cell and degassed with nitrogen for 4 nun (and for 30 s before each adsorptive stnppmg cycle). The preconcentratron potentral (usually -0.8 V) was then apphed to the electrode for a selected time, while the solutton was starred at 400 r-pm. The strmng was then stopped, and after 15 s the voltammogram was recorded by applying a negattve-going dtfferential pulse scan. The scan was termmated at - 1.5 V, and the adsorpttve stnpping cycle was repeated usmg a new mercury drop. All data were obtamed at ambrent temperature RESULTS AND DISCUSSION Ftgure 1 shows drfferentral pulse voltammograms at a statrc mercury drop electrode that had been immersed m a stirred 7.5 X lo-’ M brhrubm solutron for increasing penods of ttmc Tae longer the preconcentration tune, the more bJn-t.tbtn 1s adsorbed onto the surface and the larger the peak current For a 300 s preconcentratton penod (curve d), an 11 fold enhancement of the response 1s obtamed over that attained by conventicnal solution-phase voltammetry (0 s, curve a) Besides the

-0.9

-1.2

-1.5

E/V Fig 1 Effect of preconcentraLon penod on the tiferenual pulse stnppmg current from 7 5 X 10m8 M bhbm III 0 I M sodnun ace’xe Preconcentrahon penod (a) 0. @) 60. (c) 180, (d) 300 s St-g rate 400 rpm. preconcentratlon potential -0 8 V

64

well-defmed peak at - 1.25 V, the voltammograms show a small peak at - 1.0 V and wtth the hydrogen evolutron an irreverstble peak at - 1.45 V. that coalesces background current. Prevtous studtes deaimg wrth conventtonal pulse polarographrc measurements of btlu-ubm m baste soluuons reported the appearance of two or three peaks (6.71. These studres also attrtbuted the non-linear cahbratron curves to adsorpuon of btiuubm at the surface Adsorptton of btologtcally-related compounds, eg., heme or iron protoporphyrin, has been reported usmg mercury or carbon electrodes [10,20] in the case of heme, the adsorptton phenomenon has been exploited as an effectrve preconcentratron step m a strrppmg procedure [lo] Prelmunary work m thts laboratory [21] was undertaken usmg the oxldatron of brhrubin (to brhverdm) at carbon paste electrodes. Using phosphate buffer (pH 7.4) strong adsorptton was observed, as mdtcated from the peak obtamed at +0.45 V after transferrmg the electrode to a blauk (reactant-free) solution. However. the anodtc peak was not useful analytrcally, as It showed poor concentratlon-dependence. In contrast, the adsorptive stnppmg response at the statrc mercury drop electrode possesses srgruftcant analytrcai tltrltty as rt exlubrts a concentration dependence which IS both well defined and lughly reprodubicle. Figure 2 illustrates the response to successrve standard addttions of brhrubm, each addttion effectmg a 1 X 10e8 M mcrease m concentratron: 90 s preconcentration periods were employed Well defmed peaks are observed at the I x lo-* M-6 X lo-” M concentratton level. In contrast, the conventtonal solutron-phase dtfferenttal pulse response (dotted lures) exl-ubrts poorly defmed peaks wluch cannot be quantified conveniently_ The resultmg plot of peak current vs. concentretion (also shown rn Fig 2) IS hnear (slope 16.4 nA/lO-* M, correlatron coefficient, 0.997). Such lmearity prevails as long as linear

I

c Fig 2 Srnppq voltaniiograms obtauwd afler mcreasmg the bllwubln concentration ?111 X lo-* M steps (a-f) Preconcentrauon for 90 s at -0 8 V wth sbmng of 400 ‘pm The dotted hnes represent the duect (0 5) response

65

Isotherm condrtrons (low surface coverage) exist Frgure 3 shows the dependence of the drfferentral pulse stnpping peak current on the brln-ubm concentration using different preconcentratton times For 30 s precoccentratron (a) the response IS hnear for the entire concentratron range, 2.5 x lo-’ M-2 x 10e7 M, exammed (slope, 10.5 nA/lO-s I%, correlation coefftcrent, 0.996) At 60 and 120 s [(b) and (c)] the response IS lmear up to 1.25 x 10m7 M (slopes of the nutral hnear porhons. 17.3 and 22.2 r1AjL0-~ M, correlation coefticrents, 0.999 and 0.997, respectively) Overall, the data of Figs. 2 and 3 show that depending on the operatronal condrtrons, hqghly hnear response IS obtamed for concentratrons lower than 2 X 10m7 M (for which the adsorptive stnppmg procedure was developed). At higher concentrations, conventronal pulse voltammetry can be utrlrzed. The reproducibrhty was esttmated by ten successrve measurements on a strrred 2 X lob7 M brhrubm solutron (40 s preconcentratron) The mean peak current was 421 nA with a range of 406-442 I-IA and a relative standard devratron of 2.7%. Such precrslon compares favorably wrth that reported for other compounds measured by adsorptive stnppmg analysts [13-151. The detectabrhty of the adsorptrve stnppmg procedure IS rllus!rated u-r Frg 4 Well-defmed peak IS observed for 5 X 10m9 M brhrubrIl usmg 15 mm preconcentratron (b) In contrast (and as expected), no detectable response IS observed usmg solutton-phase pulse polarography (0 mm - a). A detectron hmit. deftned as twrce the height of the base lure npples, of 5 x lo- lo M IS estimated Detection hmrts of 3 x lob9 M and 8 X lo-” M blhrubm were obtarned using 1 and 10 mm preconcentratron penods, respectrvely. In contrast, solutron-phase Folarography results m a detectton lmut of about 5 x lo-* M (e g. Fig la) Hence, at low analyte concentration, the preconcentratron provrdes a constderably Improve? response over that obtamed by conventtonal pulse polarography To the best of our knowledge. the detectablhty obtamed m the present work (5 x lo-” M) IS the lowest reported for

300

-

200-

10-B

Cone/M

Fig. 3 Dependence of the dlfferentml pulse strippmg peak current on blhrubm ccmcen?.raLIonat d:fferent preconcenwation penods. (a) 30. (b) 60. (c) 120 s Other con&clons as III Fig 1 Dotted hnes correspond IO a lmear fit to the fist por~wn of each curve

adsorptive stnpping analysis of orgamc compounds. Detection hn-uts at the nanomolar level have been reported for adsorptive stripping measurements of other reducible species at mercury electrodes [10,15-171. Simrlar measurements of oxidizable spectes at solid electrodes usually result in higher detection hmits [9,21,15], unless longer preconcentration penods are used [14]. This is due to additional background current components that characterrze solid electrode surfaces The use of subtractive adsorptive stripping analysis can correct for these background contnbutlons 1221.Another advantage of adsorptive stripping measurements at the static mercury drop electrode is the introductton of new drop before each measurement. Thus, no “cleanmg” penod IS required at the end of the potentral scan (unlike the the use of solid electrodes where such cl eanmg is usually requtred, prolongmg analytical procedure) In contrast, the medmm exchange procedure (used for rmprovrng the selecttvrty [13]) is more eastly accomplished wrth solid electrodes. The accumuliatton of brhrubin IS affected by the mass-transport condrtrons dunng the preconcentratton step. rJsing 400 t-pm (and 120 s preconcentratton) the peak current for 2 X la-’ M bdu-ubm was 2.92 times larger than the correspondmg peak obtained wrth a qurescent (unstirred) solutron Such mass-transport control is expected, as the inherent rate of adsorption on mercury from aqueous solutton is usually fast. Delahay and Fake [23] have shown that for dtffusion-controlled adsorpnon, the quantity of adsorbed material 1s proportional to the square root of the adsorption time (as long as the frachon of the surface covered 1s far from Its equtlibnum value). Such behavtor is illustrated in Fig. 5, where the differential pulse stnppmg peak current exhrbits a square-root dependence on the preconcentratton penod for the different concentrahons of bllirubm tested As the quantity of adsorbed matenal depends on the product c!‘/‘, some devratrons from lmearity are

i

5onA

bx

i

Fig 4 Measurements of 5X10m9 M bhrubrn usmg 0 mm condiuons as UI FIN 1

(a) and 15 mm @) preconcenu&tlon_ Other

67 iB

A

1

500

y---q

I

Fig 5 Dependence of the &fferentA pulse stnppq peak current on the precuncer~tratlon pcnod at dlfferent bduubm concentrations (a) 4X 1Om8 M, (b) 8 X lo-” M, (c) 2X lo-’ M Other condmons as m FIB 1

observed (curve

when the fraction

c for large

of the surface

concentration

and

time

covered values).

approaches Statlsttcal

the equlhbnum treatment

of these

value data

gven III Table 1 Slmllar behavior wds reported recently for adsorptive stnppmg measurements of clmetldme [17]. The adsorption properties of the compound can vary with the composltlon of the supporting electrolyte. Vanous basic electrolytes, e g , phosphate buffer, borate buffer, ammomum acetate or sodium acetate, were evaluated as slutable media for the adsorptive strippmg measurement of btirubin Best resuits (with respect to signal enhancement and reproduclblhty) were obtamed m the sodmm acetate electrolyte This medium was employed throughout tlus study The adsorptive strippmg signal of bihrubln depends on the sample pH. Figure 6A shows the dependence of the peak enhancement factor (the ratio of the peak current at a gven preconcentratlon time to that wlthout preconcentratlon) on the solution pH. No response to bllirubm was observed m soiutlons more acidic than pH 6 9. Under these con&tlons, bllu-ubin 1s

TABLE

1

Dependence of drfferentml pulse stnppmg bdnubm Condmons are as m Fig. 5 Cone /M

Slope/nA

4x10-s

5 63 IS30 36 77 =

8x10-’ 2x10-7

s-‘~

a Slope of the lnltlal Iwear poruon

peak current

on preconcentrauon

penod and concentration

Intercept/nA

Correlation

0 92 -39 129

0 998 0998 0 996

coefficient

of

68

partly aggregates into nucelles and its molectiles are not avatlable for reduction [5] Increastng pH from 6.9 to 8.2 resulted m rapid increase m the peak enhancement factor. No further changes were observed over the 8.2-12 pH range. However, the stabthty of bihrubm m aqueous solutions decreases with increasing pH (above 9 [S]). Accordmgly, pH 8.2 was used throughout to sat@ the sensittvity and stability requirements The pH profile shown m Fig. 6A can be explained from the chemtcal change m the bilirubin molecule. Bthrubin is a weak base [6], and thus its ionic species tends to remam in the polar solution. (The pK for the loss of a proton from a -NH pyrrole group JS - 8.2). To test the effect of the sohttron tonic strength upon the peak enhancement factor, the sodmm acetate concentration was changed from 0 05 to 0.25 M. Essenttahy the same peak enhancement factor, - 3.6, was obtained over thrs electrolyte concentratton range (condittons: 60 s accumulatton wtth 2 starred 2 X lo-’ M bihrubin solutton) For vanous analytes, the adsorptive stnppmg response can be mfluenced by the concentrahon of :he supporting electrolyte, via saitmg-out effects [12]. Also shown tn Ftg 6 IS the dependence of the stripping peak current upon the accumtdatron potentkd (B). No sigmfrcant difference m the rate of adsorption IS observed for different potenttak over the -0.2 to -0.8 V range. To shorten the stnppmg step, a potential of -0.8 V was used throughout Practtcai apphcattons of adsorptive strippmg analysts would suffer from interferences due to the presence of surface acttve compounds. The adsorption of such compounds on the worktng electrode would mIubtt the accumulation of the analyte

iP/mo

,i,

3

t-___J - 0.2

- 0.4

-0.6

-0.8

EaccJv Fig 6 Effect of pH (A) and accumulauon potenllal(F3) on the blluubm stnppmg peak Preconcentrauon for 90 s (i) and 120 s (B) Bhrubm concentrauon. 1 x lo-’ M (A), 2~ lo-’ M (L3) Other con&!ons as m Fig 1

69

and exhibit

of the stripping signal (depressron effects are common u-r stnppmg analysis where the adsorbed film blocks the deposltton of the metals [24,25]). Knowledge of these changes IS reqmred for understandmg and minimtzing then effects The effect of gelatm (a known maximum suppressor) on the brlirubin stnpping response 1s shown III Fig. 7A Three ppm gelatm depress the depresslon

conventional

bllrrubin peak current up to 70% of its antrol

(0 ppm);

one ppm gelatin results m

25% decrease of the peak. The suppresston effect depends on the preconcentration penod (compare (a) 60 and (b) 30 s). These peak reductions are accompanied by a gradual broadening of the peak and its shift m the posrtrve drrectron. Depressron effects of surface active compounds were observed m adsorpuve stnppmg measurements of other compcunds [16,17]. From the analytical point of view, these mterferences may be corrected using the standard addition procedure Such correctton IS vahd when the percent depressron of the peak current 1s mdependent of the analyte concentrat?on (I.e., depends only on the fractton of the surface covered by the mterfermg surfactant) and the response IS hnear. For example, Frg 8 shows that the standard addttton procedure can compensate for the decrease rn the stnpping-peak currents caused by the addthon of gelatin; the response after the addrtrons of known amounts of bihrubin IS hnearly proporttonal to the brhrubm concentration. To ehmmate the effect of interfering surface active compounds, separatron by gal

Fig 7 Effect of gelatm (A) and bromde eon (B) on the blluubm (1 5x lo-’ Preconcentrauon penod. (a) 60 and (b) 30 s Other condmons as m Rg. 1

M)

stnpptng peak

70

i loo.

6

l-2 108 b"C /M

a

E Fig 8 Quant~ta~~on of bdn-ubln m t.he Presence of gelatm (a) 6x10-’ M bthrubm. (b) same as (a) but after addruon of 2 ppm gelaun. (c) same as (b) but after addmon of 6X lo-* M blhrubm, (d) same as (c) but after ad!Uon of 6x lo-* M blhrubm Preconcentrat~on time 45 s Other condltlons as m Fig 1 Also shown the resulung cahbrauon plot based on the data for (b)-(d)

frltratton can be used. For example, gel frltratton on Sephadex column was used by Kalvoda [16] pnor to adsorpttve stnppmg measurements of 5 X 10m8 M mtrazepam u-r the presence of 10 ppm gelatm. Spectftc adsot-ptton of an ran (Itke Br-) from the supporttng electrolyte can alter the double-layer capacttance and may block the electrode surface As a result, the adsorpttve stnppmg response may be changed. The effect of varymg the Brconcentration on the blhrubln stnppmg peak current IS shown II-IFig 7B A gradual decrease m the stnppmg peak IS observed (up to 40% reductron at 1 5 X lo-’ M Br-), further addrtton of Br- caused no further change The shape and posttton of the peak were not altered by the Br- addttrons In concluston, the results presented above confirm that the apphcatton of adsorpttve preconcentratlon scheme enables very sensitive voltarnmetrlc measurements of brltrubm The in srtu accumulatton step can result also tn improved selechvrty, as interferences from solutron-phase electroactrve species can be elnnmated by the medtum-exchange procedure [13] Usmg the static mercury drop electrode thrs may be best acheved m a flow system. For example, the mamfold of flow injectron systems can provrde the medmm-exchange step, as well as reproducible convechon transport dunng the accumulation 191. Chnical samples containmg surfactants would requtre pnor separatton Work m this laboratory IS contmuing m thrs dtrectron. ACKNOWLEDGEMENT

PA-M. Screnttfrc

Fanas acknowledgel, Development (CNPq)

the ftnanctal support of the Nahonal Counctl for of the Brazthan government. D.B. Luo acknowl-

71

edges the financtal support of the Untted Nattons Educattonal. Sclentlflc and Cultural Organization (UNESCO). The expenmental assistance of J.S. Mahmoud 1s appreciated. This work was supported by the NatIonal Institute of Health, Grant No GM30913-OlAl. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

20 21 22 23 24 25

B T Doumas. B W Ferry, E A Sasse and J V StraumGord, Jr, Cbn Chem , 19 (1973) J D. Vat Norman and R Szentnmay, Anal Chem ,46 (1974) 1456 C Shfstem and M Anel, J Ekctroanal Chem, 48 (1973) 447 J D Van Norman and M hl Humans, Anal Chem. 46 (1974) 926 T R Koch and 0.0 Akmgbe. Chn Chem ,27 (1981) 1295 C Shfstem and M Anel. J. Elec&oanal Chera, 75 (1977) 551 J Saar and C Yarmtzky, Isr J Chem, 23 (1983) 249 T B Jarbav and W R Hememan, Anal Chim AC@ 135 (1982) 359 J Wang and B A Frelha, Anal Chem , 55 (1983) 1285 C F Kolpm and H S Swofford, Anal Chem , 50 (1978) 916 J W Su-m and RP Baldwm. Anal I.&L.. 13 (1980) 577 R Kalvoda, Anal Chlm Acta. 138 (1982) 11 J. Wang and BA. Frelha, Anai Chun. Acta. 148 (1983) 79 H Y Cheng, L Falat and R L LI, Anal Chem , 54 (1982) 1384 EN Chaney and RP Baldwm. Anal Chem , 54 (1982) 2556 R Kalvoda, Anal Chm~ Acta, 162 (1984) 197 A Webber. M Shah and J. Osteryoung, AnaL Chun Acta, 154 (1983) 105. H W Nlrmberg Pure Appl Chem ,54 (1982) 853. H Sawamoto. J Ekctroanal Chem , 147 (1983) 279 A P Brown, C Koval and F C Anson. J Electroanal Chem . 72 (1976) 379. L Hutchms and 3 Wang, unpubbhed results J Wang and BA Frelha, Talanta. 30 (1983) 837 P Delahay and C F&e, J Am Chem Sot , 80 (1958) 2628 P L Brezomk, P_A Brauner and W Stumm, Water Res , 10 (1976) 605 J Wang and D B Luo. Talanta, 31 (1984) 703

984