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Page 1: Determination of tumor marker CA125 by capillary electrophoretic enzyme immunoassay with electrochemical detection

Analytica Chimica Acta 497 (2003) 75–81

Determination of tumor marker CA125 by capillaryelectrophoretic enzyme immunoassay with

electrochemical detection

Zhihui He1, Ning Gao, Wenrui Jin∗Laboratory of Analytical Science, School of Chemistry and Chemical Engineering,

Shandong University, Jinan 250100, China

Received 24 March 2003; received in revised form 10 June 2003; accepted 11 July 2003

Abstract

A novel capillary electrophoretic enzyme immunoassay with electrochemical detection (CE-EIA-ED) was developed fora tumor marker cancer antigen 125 (CA125). In this method, after the noncompetitive enzyme immunoreaction, the freeenzyme (horseradish peroxidase)-labeled anti-CA125 antibody (Ab∗) and the bound enzyme-labeled complex (Ag–Ab∗) wereseparated in a separation capillary and then catalyzed the enzyme substrate (3,3,5,5-tetramethyl-benzidine dihydrochloride,TMB(Red)) and H2O2 in a reaction capillary following the separation capillary. The product of the enzymatic catalysisreaction (TMB(Ox)) was amperometrically detected on a carbon fiber microdisk bundle electrode. A activity concentrationlimit of detection (LOD) of 0.29 U/ml, which corresponded to a activity LOD of 1.6�U was achieved. The assay could beused to determine CA125 in human serums from ovary tumor patents.© 2003 Elsevier B.V. All rights reserved.

Keywords: Capillary electrophoresis; Electrochemical detection; Immunoassay; Tumor marker

1. Introduction

Cancer antigen 125 (CA125), an ovarian cancer as-sociated antigen, is defined by the murine monoclonalantibody OC125, which was obtained by Bast and hiscolleagues using the ovarian cell line OVCA433 asimmunogen[1]. The antigen is located on the surfaceof ovarian tumor cells, with restricted expression innormal adult tissues such as endocervix, endometrium,tubes, pleura, pericard, peritoneum, and occasionalexpression in intestine, lung and kidney[2–4]. In sera

∗ Corresponding author. Fax:+86-531-8565167.E-mail address: [email protected] (W. Jin).

1 Present address: Technical Center of Changde CigaretteFactory, Changde 415000, China.

of patients with carcinoma, the CA125 antigen is notexclusive to ovarian carcinoma, but is shown to beelevated in a large number of different cancers[5,6].The CA125 antigen has also been shown to be presentin breast milk (particularly colostrum), ascites, cystfluid, cervical secretion, uterine secretion and amni-otic fluid [7–10]. CA125 is an antigen present on 80%of nonmucinous ovarian carcinomas. It circulates inthe serum of patients with ovarian carcinoma and istherefore investigated for possible use as a marker. Forhealthy human, the concentration levels of CA125 arelower than 35 U/ml[11,12]. Assays for CA125 havegreat clinical importance. In clinic research, CA125levels are often measured by immunoradiometricassay [11], enzyme immunoassay[12–15]. Theseconventional immunoassay have some shortcoming

0003-2670/$ – see front matter © 2003 Elsevier B.V. All rights reserved.doi:10.1016/S0003-2670(03)00880-8

Page 2: Determination of tumor marker CA125 by capillary electrophoretic enzyme immunoassay with electrochemical detection

76 Z. He et al. / Analytica Chimica Acta 497 (2003) 75–81

such as time-consuming (ca. 4 h), high reagentconsumption (ca. 5�l) and complicated operation(over 10 steps).

Capillary electrophoresis (CE) is a powerful tech-nique for the separation of macromolecules such asproteins and immunocomplexes[16]. With both supe-rior separation power and high detection sensitivity,CE can separate free antibody or antigen from boundantibody or antigen rapidly, and is especially suitablefor immunoassay[17]. The method called capillaryelectrophoretic immunoassay (CEIA) offers severaladvantages such as short analysis time (ca. 1.5 h), lowreagent consumption (less 1�l), and simple opera-tion (ca. two steps) over conventional immunoassays.The procedure of immunoassay can be simplified byCE separation and many wash steps can be elim-inated. In CEIA, UV detection and laser-inducedfluorescence (LIF) detection have been used. How-ever, the major disadvantage of the UV detectionis the lack of sensitivity. The minimum detectableconcentration by UV detection is around 10−6 mol/l.LIF detection is a more general approach to improvesensitivity.

Amperometric detection provides excellent sensi-tivity for the small dimensions associated with CE,while offering a high degree of selectivity towardselectroactive species. In our present work, a capillaryelectrophoretic enzyme immunoassay with electro-chemical detection (CE-EIA-ED) using a noncompeti-tive format has been developed and applied to monitorCA125 in serum. In the assay, an excess amount ofhorseradish peroxidase (HRP)-labeled anti-CA125 an-tibody, Ab∗, is added to the sample to form its boundcomplex, Ag–Ab∗, with the antigen CA125 (Ag)present in the sample. Then, Ab∗ and Ag–Ab∗ are sep-arated by CE in the separation capillary. Both enter thereaction capillary following the separation capillaryand catalyze the reaction of enzyme substrate, reducedform of 3,3,5,5-tetramethyl-benzidine (TMB(Red))and H2O2. The reaction product, oxidized form of3,3,5,5-tetramethyl-benzidine (TMB(Ox)), is amper-ometrically detected at a carbon fiber microdisk bun-dle electrode at the outlet of the reaction capillary.Since the concentration of TMB(Ox) is much higherthan those of the free Ab∗ and the Ag–Ab∗ due tothe enzyme amplification, the activity concentrationlimit of detection (LOD) (3σ) of CA125 is as lowas 0.29 U/ml (or a activity LOD of 1.6 × 10−6 U).

The assay was used to determine CA125 in humanserum.

2. Experimental

2.1. Apparatus

The CE-EIA-ED system used here was the sameas in our previous description[18]. Briefly, it con-sisted of the three parts: a polyacrylamide-coatedseparation capillary, a polyacrylamide-coated reac-tion capillary following the separation capillary anda electrochemical detection system. A high-voltagepower (Model 9323-HVPS, Beijing Institute of NewTechnology, Beijing, China) provided a variable volt-age of 0–30 kV across the separation capillary (50�mID, 375�m OD, 15 cm length), with its outlet endat ground potential. The combination of the separa-tion capillary and the reaction capillary (50�m ID,375�m OD, 5 cm length) was similar to a post-columnreactor. The enzyme substrate (TMB(Red)) solu-tion was introduced into the reaction capillary bymeans of a liquid pressure. The enzyme-catalyticproduct TMB(Ox) eluting from the reaction capil-lary was determined by the electrochemical detectionsystem.

The reaction capillary and the detection cell werehoused in a Faraday cage in order to minimize theinterference from noise of external sources. ED at aconstant potential was performed with the electro-chemical analyzer (Model CHI800, CH Instrument,Austin, TX). ED was carried out with a three-electrodesystem. It consisted of a carbon fiber microdisk bun-dle electrode as the working electrode, a saturatedcalomel electrode (SCE) used as the reference elec-trode, and a coiled Pt wire (0.3 mm diameter, 5 cmin length) placed at the bottom of the cell as theauxiliary electrode. The carbon fiber microdisk bun-dle electrodes used here were described previously[19]. Before use, all carbon fiber microdisk bundleelectrodes were cleaned in alcohol and washed withdouble-distilled water for 5 min by an ultrasonicator.

2.2. Capillary treatment

The polyacrylamide-coated capillaries were pre-pared from the uncoated fused-silica capillaries with

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Z. He et al. / Analytica Chimica Acta 497 (2003) 75–81 77

50�m ID. The inner surface of capillaries was firstpretreated with 1 mol/l NaOH for 30 min and thenflushed with water for 30 min. The silane solutionadjusted to pH 3.5 by acetic acid containing 0.5%(v/v) �-methacryloxypropryltrimethoxysilane (AcrosOrganics, New Jersey) and 0.5% (v/v) alcohol wassucked up into the capillaries. After reaction pro-ceeded for 1 h at room temperature, the silane solutionwas removed. Then the capillaries were filled with3.5% (w/v) deaerated acrylamide solution containing1�l N,N,N′,N′-tetramethylethylenediamine (TEMED)and 2 mg potassium persulphate per milliliter. After3 h, the excess (not attached) polyacrylamide wassucked away and the capillaries were rinsed withwater. After most of the water in the capillaries wasremoved by aspiration, they were then dried under aN2 stream at 45◦C.

2.3. Immunoassay procedure

The immunoassay protocol was a noncompetitiveformat. A 25�l aliquot of the CA125 standards, orserum samples and a 5�l aliquot of HRP-labeledanti-CA125 antibody were added to a microcen-trifuge tube. The solution was incubated for 1 h atroom temperature, and then was diluted to 150�lwith the running buffer. Then hydrodynamic injectionwas performed with a 9 cm height for 20 s. A sepa-ration high-voltage was applied across the separationcapillary and the detection potential was applied atthe working electrode. When the separated Ab∗ andAg–Ab∗ ran from the separation capillary into thereaction capillary, whereupon both catalyzed the reac-tion of enzyme substrate TMB(Red) and H2O2. Thereaction product, TMB(Ox), was detected at the outletof the reaction capillary. In the electrochemical de-tection, the working microdisk bundle electrode wascemented onto a microscope slide, which was placedover a laboratory-made XYZ micro-manipulator andglued in place in such a way that the microdisk endprotruded from the edge of the slide. The position ofthe microdisk bundle electrode was adjusted (undera microscope) against the outlet of the reaction cap-illary so that the electrode and the capillary were incontact. This arrangement allowed easy removal andrealignment of both the capillary and the electrode.All potentials were measured against SCE. All dispos-able plastic wares and disposable micro-pipette tips

used in the assay were autoclaved prior to use in or-der to denature any contaminants. All solutions wereprepared in disposable plastic ware using disposablepipette tips.

2.4. Reagents

The CA125 EIA kit (no. 400-10) was purchasedfrom CanAg Diagnostics AB, Gothenburg, Sweden,which consisted of CA125 standards (containing 0,10, 40, 200 and 500 U/ml), and a solution contain-ing two mouse monoclonal anti-CA125 antibodies(Ov197 and Ov185) (30�g/ml) labeled with HRP.The kit was stored at 4◦C. The ovary cancer serumsamples and the results detected by ELISA were pro-vided by Hematological Center, Qilu Hospital, Jinan,China. The serum samples were stored at−20◦C.TMB(Red) (High Pure Grade) was obtained fromAmresco Inc. (Solon, OH). A stock standard so-lution of TMB(Red) (0.020 mol/l) was prepared indouble-distilled water and kept in a dark bottle. Therunning buffer consisted of 2.0 × 10−3 mol/l H2O2,2.5 × 10−4 mol/l Na2B4O7 and 9.0 × 10−3 mol/lH3BO3 (pH 7.4). The substrate solution consistedof 2.0 × 10−4 mol/l TMB(Red), 1.0 × 10−2 mol/lNa2HPO4 and 5.0 × 10−3 mol/l citric acid (pH 5.0).TMB(Red) or H2O2 was added to the buffers justbefore the measurement. The running buffer wasrenewed every run. All buffers and solutions werestored at 4◦C until use. Unless stated otherwise, allother reagents were of analytical grade and purchasedfrom standard reagent suppliers. All solutions wereprepared with double-distilled water. All buffers werefiltered through 0.45�m cellulose acetate membranefilters (Shanghai Yadong Resin Co. Ltd., Shanghai,China) before use.

3. Results and discussion

3.1. Optimization of CE-EIA-ED

In the assay, the immunoassay protocol is a non-competitive format. The separated Ab∗ and Ag–Ab∗catalyze the reaction of the substrates TMB(Red)introduced into the reaction capillary by a liquid pres-sure and H2O2 in the running buffer. The catalysis

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78 Z. He et al. / Analytica Chimica Acta 497 (2003) 75–81

reaction is shown as follows[20]:

The enzymatic reaction product TMB(Ox) can be re-duced at the carbon fiber microdisk bundle electrodeaccording to the following scheme[20].

Both Ab∗ and Ag–Ab∗ can be detected throughmeasuring TMB(Ox) at the outlet of the reactioncapillary. In our experiments, 1.0 × 10−2 mol/lNa2HPO4–5.0 × 10−3 mol/l citric acid (pH 5.0)containing 2.0 × 10−4 mol/l TMB(Red) buffer wasused as the substrate. To obtain the high and ran-now electrophoretic peak of Ab∗, different runningbuffers of pH 7.4 (Na2HPO4–citric acid, Tris–HCl,Na2B4O7–H3BO3) were tested. It is found that2.5 × 10−4 mol/l Na2B4O7–9.0 × 10−3 mol/l H3BO3of pH 7.4 is better as the running buffer. The mi-gration time, tm, the peak area,q, the width atthe half-peak,W1/2, and the number of theoreticalplates,N, of Ab∗ at different concentrations of H2O2,CH2O2, are listed inTable 1. tm and N are almostconstant. WhenCH2O2 < 1.0 × 10−3 mol/l, q in-creases rapidly with increasingCH2O2. WhenCH2O2

is between 1.0 × 10−3 mol/l and 2.0 × 10−3 mol/l,q is almost a constant, which indicates the satu-ration of H2O2 for HRP-labeled. However, whenCH2O2 > 2.0 × 10−3 mol/l, q decreases with increas-ing CH2O2. This is because the activity of HRP is lesssensitivity to excess H2O2 [21]. The maximumq wasobtained using 2.0 × 10−3 mol/l H2O2. The valuewas used for determination of CA125. It is possible

that H2O2 reacts with the capillary and the carbonfiber electrode in a reproducible manner because ofgood reproducible results. Therefore, H2O2 did notaffect the results, when the product TMB(Ox) wasdetected.Fig. 1 shows the relationship betweenq andthe applied detection potential,Ed. WhenEd is morepositive than 0.10 V,q increases with decreasingEd.WhenEd is more negative than 0.10 V,q is almost a

Table 1The values oftm, q, W1/2 and N at different concentrations ofH2O2 in the running buffer,CH2O2 (running buffer, 2.5×10−4 mol/lNa2B4O7–9.0×10−3 mol/l H3BO3 (pH 7.4); 1.0�g/ml Ab∗; sub-strate solution, 2.0 × 10−4 mol/l TMB(RED) in 1.0 × 10−2 mol/lNa2HPO4–5.0 × 10−3 mol/l citric acid (pH 5.0); separation cap-illary, 25 cm× 50�m ID; reaction capillary, 5 cm× 50�m ID;hydrodynamic injection, 9 cm for 20 s; separation voltage, 20 kV;detection potential, 0.00 V vs. SCE)

CH2O2

(10−3 mol/l)tm (min) q (nC) W1/2 (s) N (104)

0.2 12.2 0.85 8.3 4.30.5 12.1 1.15 8.5 4.01.0 12.0 2.35 8.6 3.92.0 11.8 2.40 8.6 3.85.0 11.6 1.85 8.4 3.8

10 11.5 1.03 8.2 3.9

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Z. He et al. / Analytica Chimica Acta 497 (2003) 75–81 79

0.5 0.4 0.3 0.2 0.1 0.0 -0.1 -0.2

0.8

1.2

1.6

2.0

2.4

2.8

q (

nC)

Ed (V vs SCE)

Fig. 1. Relationship between the detected electric charge,q, and thedetection potential,Ed; 2.0×10−3 mol/l H2O2 and other conditionsare same as inTable 1.

constant.Ed of 0.00 V was used because of largerq andlower noise.tm, q, W1/2, andN at different separationvoltages,Vs, are listed inTable 2. From Table 2, itcan be seen thattm andW1/2 decrease andN increaseswith increasingVs. This is because higher values ofVs give higher electroosmotic flow rate. WhenVs <

20 kV, q is a constant because of identical coulometricefficiency. However, whenVs > 20 kV, q reduces withincreasingVs. In this case,tm shortened and only a partof TMB(Ox) can be reduced at the working electrode,i.e. the coulometric efficiency is decreased. Therefore,20 kV for Vs was chosen in our experiments.

3.2. CE-EIA-ED for CA125

In this method the noncompetitive format was per-formed. CA125 (Ag) reacted with an excess amountof the solution containing two HRP-labeled mono-clonal anti-CA125 antibodies (Ab∗) from the EIA kit.

Table 2The values ofq, tm, W1/2 and N at different separation voltages,Vs (2.0 × 10−3 mol/l H2O2 and other conditions are same as inTable 1)

Vs (kV) tm (min) q (nC) W1/2 (s) N (104)

10 13.1 2.40 17.8 1.115 12.6 2.41 10.5 2.918 12.2 2.36 8.8 3.820 11.8 2.40 8.6 3.822 11.5 1.88 8.0 4.125 11.1 0.90 6.9 5.2

After the noncompetitive reaction was completed, thesolution containing Ab∗ and Ag–Ab∗ was injectedinto the separation capillary and Ab∗ and Ag–Ab∗were separated by CE in the separation capillary. Bothcatalyzed TMB(Red) and H2O2 in the reaction cap-illary following the separation capillary. The reactionproduct, TMB(Ox), was detected at the outlet of thereaction capillary on the carbon fiber microdisk bun-dle electrode. Thus, two peaks corresponding to Ab∗and Ag–Ab∗ should appear in the electropherograms.The electropherograms obtained are shown inFig. 2

Fig. 2. Electropherograms of the solutions for different concentra-tions of CA125. Concentration of CA125 (U/ml): (1) 0; (2) 1.67;(3) 6.67; (4) 33.3; (5) 83.3; 2.0 × 10−3 mol/l H2O2 and otherconditions are same as inTable 1.

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80 Z. He et al. / Analytica Chimica Acta 497 (2003) 75–81

Table 3Results detected and recovery of CA125 in serum samples (2.0 × 10−3 mol/l H2O2 and other conditions are same as inTable 1)

Sample Determined value (U/ml) Average value (U/ml) Added value (U/ml) Observed value (U/ml) Recovery (%)

I 23.5 24.5 13.3 37.2 10324.7 20.0 43.5 9425.3 25.0 49.8 98

II 35.3 34.7 13.3 49.5 10734.2 25.0 58.5 9734.7 33.3 66.8 96

at different concentrations of CA125. With increasingthe concentration of CA125,q of peak 1 decreasesandq of peak 2 increases. According to the principleof the noncompetitive assay, peaks 1 and 2 should bethe peak of Ab∗ and the peak of Ag–Ab∗, respectively.Although the post-capillary catalysis reactor and theend-capillary amperometric detector could introducethe post-capillary zone broadening, the enough res-olution for the both peaks did not affect their mea-surement. It can be found fromFig. 2 that the peak1 (the peak of Ab∗) consists of two peaks. The twomonoclonal antibodies in the EIA kit should be re-sponsible for that. It was also verified by the fact thatpeak 2 (the peak of Ag–Ab∗) was obviously dividedinto two peaks, when the concentration of CA125 wasincreased. Therefore, the total peak area of the peak2 was used for quantification of CA125. The calibra-tion curve based on the peak of Ag–Ab∗ is shown inFig. 3. The points represent the averageq detected

0 20 40 60 800.0

0.3

0.6

0.9

1.2

1.5

q (

nC)

CCA125 (U/mL)

Fig. 3. Calibration curve based on the total peak area of thecomplex of CA125 with its antibody, Ag–Ab∗. Running buffer,2.5×10−4 mol/l Na2B4O7–9.0×10−3 mol/l H3BO3 (pH 7.4) con-taining 2.0 × 10−3 mol/l H2O2 and other conditions are same asin Table 1.

for three runs. LOD for the noncompetitive assaycalculated by using the meanq of the Ag–Ab∗ peakfor the zero-dose CA125 plus three times its standarddeviation calculated from 10 trials was 0.29 U/ml. Ac-cording to Hagen–Poiseuille equation, the injectionvolume calculated was 5.5 nl for the hydrodynamic in-jection with 9 cm height for 20 s. Therefore, a activityLOD of 1.6 × 10−6 U can be obtained. The responsefor a series of six injections of 6.67 U/ml CA125 re-sulted in a relative standard deviation of 4.8% fortmand 3.9% forq. In order to verify the method, twoserum samples from different ovary tumor patientswere detected according to the procedure describedin the experimental section. The determined results ofthe diluted samples are shown inTable 3. The concen-trations of CA125 in the two samples obtained by thecalibration curve are 139 and 201 U/ml, respectively.Also, the two serum samples were determined byHematological Center, Qilu Hospital (Jinan, China)using the routine ELISA for comparison. The valuesof CA125 were found to be 150 and 209 U/ml, re-spectively, by ELISA. In order to prove the reliabilityof the method, a certain amount of standard CA125was added to the two serum samples. Then, the serumsamples with the standard CA125 were measured.Thus, we can obtain the recovery. The results are listedin Table 3. The determined recovery of the methodwas between 94 and 107%. Obviously, the presentCE-EIA-ED is a simpler and more time-saving.

4. Conclusion

The developed CE-EIA-ED of CA125 with the non-competitive format is a new useful method with highselectivity, low LOD and low sample consumption. Itshould be noted that this approach is not limited to

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Z. He et al. / Analytica Chimica Acta 497 (2003) 75–81 81

the determination of CA125. In many commerciallyavailable enzyme immunoassay kits, HRP is labeledon antigen or antibody, and the TMB(Red) is used asenzyme substrate. Therefore, CE-EIA-ED with a non-competitive format based on the catalysis action ofHRP can easily be used to determine other antigens orantibodies. This method is useful where the HRP en-zyme label is available and a fluorescent label is not.We think that CE-EIA-ED will become a useful toolin immunological assays.

Acknowledgements

This project was supported by the National NaturalScience Foundation of China (no. 20235010) and theState Key Laboratory of Electroanalytical Chemistry,Changchun Institute of Applied Chemistry, ChineseAcademy of Sciences.

References

[1] R.C. Bast Jr., M. Feeney, H. Lazarus, L.M. Nadler, R.B.Colvin, R.C. Knapp, J. Clin. Invest. 68 (1981) 1331.

[2] R.C. Bast Jr., T.L. Klug, E.St. John, E. Jenison, J.M. Niloff,H. Lazarus, R.S. Berkowitz, T. Leavitt, C.T. Griffiths, L.Parker, V.R. Zurawski Jr., R.C. Knapp, N. Engl. J. Med. 309(1983) 883.

[3] E.J. Nouwen, P.G. Hendrix, S. Dauwe, M.W. Eerdekens, M.E.De Broe, Am. J. Pathol. 126 (1987) 230.

[4] V.R. Zurawski Jr., H.M. Davis, N.J. Finkler, C.L. Harrsion,R.C. Bast Jr., R.C. Knapp, Cancer Rev. 11–12 (1988) 102.

[5] K. Kuzuya, M. Nozaki, T. Chihara, Nippon Sanka FujinkaGakkai Zasshi 38 (1986) 949.

[6] P. Kenemans, C.A. Yedema, G.G. Bon, S. von Mensdorff-Pouilly, Eur. J. Obstet. Gynecol. Reprod. Biol. 49 (1993) 115.

[7] J.M. Niloff, R.C. Knapp, E. Schaetzl, C. Reynolds, R.C. BastJr., Obstet. Gynecol. 64 (1984) 703.

[8] T.J. O’Brien, J.W. Hardin, G.A. Bannon, J.S. Norris, J.G.Quirk Jr., Am. J. Obstet. Gynecol. 155 (1986) 50.

[9] F.-G. Hanisch, G. Uhlenbruck, C. Dienst, M. Stottrop, E.Hippauf, Eur. J. Biochem. 149 (1985) 323.

[10] G.J. Fleuren, M. Nap, J.G. Aalders, J.B. Trimbos, H.W.A.De Bruijn, Cancer 60 (1987) 2437.

[11] X. Li, X. Wu, Y. Ni, Clinical Determination of TumorMarkers, People’s Healthy Press, Beijing, 1996, p. 75.

[12] J.T. Wu, T. Miya, J.A. Knight, D.P. Knight, Clin. Chem. 34(1988) 1853.

[13] J. Reinsberg, B. Schultes, U. Wagner, D. Krebs, Clin. Chem.39 (1993) 891.

[14] K.W. Ryder, T.O. Oei, M.T. Hull, M.M. Sample, Clin. Chem.34 (1988) 2513.

[15] J.O. Kang, W.A. Hudak, N. Keller, B.S. Criswell, Clin. Chem.34 (1988) 1983.

[16] P.D. Grossman, J.C. Colburn, H.H. Lauer, R.G. Nielsen, R.M.Riggin, G.S. Sittampalam, E.C. Rickard, Anal. Chem. 61(1989) 1186.

[17] J.J. Bao, J. Chromatogr. B 699 (1997) 463.[18] M. Jia, Z. He, W. Jin, J. Chromatogr. A 966 (2002) 187.[19] W. Jin, D. Yu, Q. Dong, X. Ye, J. Chromatogr. Sci. 38 (2000)

11.[20] G. Volpe, D. Compangnone, R. Draisci, G. Palleschi, Analyst

123 (1998) 1303.[21] N. Hoshio, R. Nakajima, I. Yamazaki, J. Biochem-Tokyo 102

(1987) 785.