Zak K. Shihabi

Department of Pathology,Wake Forest University Schoolof Medicine,Winston-Salem, NC, USA

Received January 19, 2006Revised March 4, 2006Accepted March 20, 2006

Research Article

Direct analysis of hydrogen peroxide bycapillary electrophoresis

A method is described for analysis of hydrogen peroxide directly by CZE in boratebuffer based on its absorption in UV light at 185 nm, without reaction with dyes. Theabsorption at 185 nm was about 3.5 times better than that at 214 nm. Hydrogen per-oxide was generated enzymatically from glucose in aqueous solutions and in serumand was removed by the catalase enzyme. To improve the sensitivity of detection,samples were concentrated on the capillary based on stacking by ACN. The method israpid (,7 min) and specific.

Keywords: Catalase / Oxidase / Oxidative stress / Oxygen-reactive speciesDOI 10.1002/elps.200600034

1 Introduction

Hydrogen peroxide is a very common compound in naturewith numerous biological and industrial applications. Insupermarkets it is sold for household use as a 3% watersolution as a cleaning solvent, and as a mild-bleachingagent. In drug stores it is available as an antiseptic, as a hair-bleach, and as a mouthwash. In industry it is used as astrong oxidizing agent and a bleaching agent for textiles(wool and silk), and also for water/waste and effluent treat-ment. Most of the hydrogen peroxide is used industrially inpaper manufacturing. Hydrogen peroxide is involved in thefamous Fenton’s reaction which is often utilized for detox-ification of many pollutants and toxins. It is also used in foodas a sterilization and spoilage control agent. It kills, orseverely inhibits, the growth of anaerobic organisms. A35% food-grade has been marketed as hydrogen peroxidetherapy. Advocates of the product claim that it can be dilut-ed and used for “hyperoxygenation therapy” to treat AIDS,cancer, colds, and many other conditions.

Hydrogen peroxide is produced in many biological reac-tions. In the living cells, hydrogen peroxide exerts bothdesirable and also harmful effects. Cells produce hydro-gen peroxide in many metabolic reactions and at thesame time they have different enzymes for its removal.Aerobic dehydrogenase enzymes characteristically pro-duce hydrogen peroxide. One main source of its produc-

tion is the superoxide dismutase in the mitochondria ofthe cell. However, it is removed rapidly in the cell by sev-eral enzymatic reactions especially the catalase and per-oxidase. Both hemoglobin and myoglobin have peroxi-dase-like activity. Hydrogen peroxide is a part of thereactive oxygen species and the oxidative stress whichcan damage proteins, DNA and especially lipids. In con-trast, neutrophils produce hydrogen peroxide as the firstline of defense against toxins, parasites, bacteria, viruses,and yeasts. Hydrogen peroxide can act as a membranereceptor signaling in cell apoptosis [1].

Many oxidase enzymes from yeast or of microbial origingenerate hydrogen peroxide on the corresponding sub-strate. Commercially these enzymes are isolated, puri-fied, and utilized to measure clinically many substratesin the serum. Currently, they are used extensively inclinical chemistry laboratories on the automated analy-zers to analyze many natural compounds present inserum and urine such as glucose, cholesterol, ethanol,triglycerides, and uric acid by the corresponding oxi-dase enzyme. These enzymes offer specificity and,moreover, do not require harsh conditions for reaction,so they have found widespread use in the automatedinstruments. Many of the common ELISAs use glucoseoxidase as a color-indicator (generating) enzyme withthe production of hydrogen peroxide. This in turn isreacted with different dyes. Furthermore, low con-centrations of hydrogen peroxide can be found in manynatural sources such as oceans, rain water, and honey.It is often present as a by-product in pharmaceuticalsduring manufacturing, and it can affect the chemicalstability of drugs in formulations.

Correspondence: Professor Zak K. Shihabi, Department of Pathol-ogy, Wake Forest University School of Medicine, Winston-Salem, NC27157, USAE-mail: zshihabi@wfubmc.eduFax: 11-336-716-9944

Electrophoresis 2006, 27, 4215–4218 4215

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4216 Z. K. Shihabi Electrophoresis 2006, 27, 4215–4218

Several techniques are used to measure hydrogen per-oxide indirectly. Most of these employ the oxidation ofdifferent dyes using colorimetric, fluorescent, or chemo-luminescent assays in the presence of a peroxidase en-zyme as an indicator step [2–8]. All these methods havedifferent degrees of sensitivity but in general they aresubject to interferences especially from other peroxidesand different reducing substances such as ascorbic acid[9, 10]. Hydrogen peroxide is an electrochemically activecompound. This property has been used successfully inconjunction with CZE to detect several peroxides includ-ing hydrogen peroxide [11, 12]. The method is very sen-sitive; however, the instrumentation is not commerciallyavailable.

Hydrogen peroxide absorbs in the low UV light (220–185 nm). The absorption of hydrogen peroxide in the UVregion is not very strong. Stacking improves the detectionin CE [13]. Several methods have been described forstacking [13]. ACN stacking is useful for samples whichcontain high protein and high salts such as serum [14].This method removes the proteins and at the same timeconcentrates hydrogen peroxide on the capillary directlythrough stacking by ACN based on transient pseudo-ITP[14].

Here, we show that hydrogen peroxide can be analyzeddirectly and specifically by CE using commercially avail-able instruments based on its absorption in the low regionof the UV light (214–185 nm). The advantage of thismethod is that hydrogen peroxide can be detected di-rectly without interferences. The method is rendered moresensitive through stacking by ACN.

2 Materials and methods

2.1 Chemicals and buffers

Glucose oxidase and catalase were purchased fromSigma Chemicals (St. Louis, MO, USA). Hydrogen per-oxide, AR, was purchased from Mallinckrodt (St. Louis,MO, USA). Boric acid (170 mmol/L) adjusted to pH 8.6with 2 mol/L sodium hydroxide. Na2HPO4 and NaH2PO4

were mixed to prepare a stock buffer of 150 mmol/L,pH 6.0 for glucose oxidase.

2.2 Instrument

Quanta 4000 (Waters, Milford, MA, USA) equipped withuntreated capillary 32 cm650 mm id. The voltage was setat 10.5 kV and the wavelength at 185 nm. The samplewas injected for 10 s under nonstacking conditions andfor 100 s under stacking conditions and was electro-

phoresed for 8 min. The capillary was washed betweensamples for 1.5 min with 1 mol/L sodium hydroxide andflushed with electrophoresis buffer for 1.5 min.

2.3 Dialysis

Serum (1 mL) was dialyzed using C membrane, 1000 cut-off, against 1 mL of phosphate buffer, 30 mmol/L, pH 6.0in a special dialysis cell (1 mL volume) which can hold fivesamples at the same time (Science ware, Pequannock,NJ, USA), as described earlier [15]. The dialysis cells werekept rotating for 2 h.

2.4 ACN deproteinization

Serum (100 mL; containing hydrogen peroxide) was mixedwith 200 mL of ACN. The samples were mixed for 10 s andcentrifuged at 12 000 rpm for 15 s [14]. The supernatantwas injected on the capillary.

3 Results and discussion

Hydrogen peroxide absorbs in the low region of UV light(220–185 nm) without a specific maximum, and migratesas a weak anion in CZE in the borate buffer. The absorb-ance at 185 nm is about 3.5 times better than at a morecommon wavelength of 214 nm. It emerges in about6 min at pH 8.6. Below pH 8.3, the peak comigrates withan unknown system peak (Fig. 1). As the pH increases,the two peaks separate from each other but the migrationincreases too; however, the peak height decreases. Abuffer with pH 8.6 offers a compromise of good sensitivityand short migration time while separating the hydrogenperoxide from the system peak (Fig. 1). The system peakis useful as it serves as an internal standard especially forthe migration time.

Here, glucose oxidase is used as an example to generatehydrogen peroxide by reaction with glucose in vitro(Fig. 2). A small peak corresponding to hydrogen peroxideis observed after 30 min of incubation (Fig. 2B) andincreases in size with time. After 3 h of incubation, thepeak becomes large (Fig. 2C). However, the peak dis-appears rapidly upon the addition of the enzyme catalase,Fig. 2D indicating that the peak is truly that of hydrogenperoxide. It is interesting to note that the enzyme glucoseoxidase also shows as a peak in this method (Fig. 2A).Thus, glucose oxidase can be also measured after opti-mization and validation.

One of the main challenges is to be able to measurehydrogen peroxide production in complex samples suchas serum, cell extracts, or industrial samples. In the first

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Figure 1. (Top) Hydrogen peroxide (H) standard 1 g/L inwater and (bottom) blank (I, system peak).

Figure 2. Production of hydrogen peroxide (H) by theglucose oxidase reaction (G). (A) Glucose (1 g/dL) 1 glu-cose oxidase (1 mg/mL) at 0 time, (B) at 30 min, (C) 3 h,and (D) after addition of 1 m/mL of catalase (I = systempeak).

attempt, glucose oxidase was added to the serum andthe samples were incubated and dialyzed against weakphosphate buffer to separate hydrogen peroxide from thelarge molecules especially serum of proteins. Because ofits small molecular weight and rapid diffusion hydrogenperoxide was detected on the opposite side of the dialy-zer membrane (Fig. 3). However, dialysis requires a longtime (,2 h) and extra steps but it is very efficient in iso-lating serum proteins which can ruin the capillary.

Because the absorption of hydrogen peroxide in the UVregion is not very strong, a different approach was under-taken to remove serum proteins and at the same time toconcentrate hydrogen peroxide on the capillary directlythrough stacking by ACN based on transient-pseudo-ITP[12] (Fig. 4A). A serum sample containing 310 mg/dL glu-cose was mixed and incubated with glucose oxidase togenerate hydrogen peroxide. After 2 h of incubation, ACNwas added to the sample to precipitate the proteins andinduce the stacking. The supernatant was injected and alarge peak of hydrogen peroxide was detected (Fig. 4C),which was absent in the sample incubated without glu-

Figure 3. Production of hydrogen peroxide (H) fromserum: Serum (1000 mL; glucose 500 mg/dL) was mixedwith 200 mL of phosphate buffer (pH 6.0) and 1 mg glu-cose oxidase and dialyzed against 1 mL of phosphatebuffer 30 mmol/L, pH 6.0. Top, after 2 h in the absence ofglucose oxidase; bottom: in the presence of glucose oxi-dase (I = system peak).

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4218 Z. K. Shihabi Electrophoresis 2006, 27, 4215–4218

Figure 4. Stacking of hydrogen peroxide (H) generated inserum by ACN. (A) Hydrogen peroxide, standard 60 mg/Ldissolved in one volume of 1% NaCl and two volumes ofACN. (B) Serum (1000 mL; 310 mg/dL glucose) was mixedwith 200 mL of phosphate buffer (pH 6.0) in the absence ofglucose oxidase. (C) Same serum sample in the presenceof 1 mg/mL of glucose oxidase; injection 90 s (I = systempeak).

cose oxidase (Fig. 4B). This stacking gives about 10–15-fold increase in concentration compared with directinjection from water.

The analysis from aqueous solutions was linear between60 and 600 mg/L (r = 0.995, n = 5). The minimum detec-tion level by ACN is 6 mg/L (36 baseline noise). The RSDfor ten analyses was 2.6% for peak height and 1.7% formigration time (n = 10).

In spite of the stacking, the hydrogen peroxide has muchbetter sensitivity (LOQ) by electrochemical detection [11,12]. Although the CE step offers specificity, the electro-chemical detection through appropriate selection of thereduction/oxidation potential adds another layer of spec-ificity to the analysis. The electrochemical detector candetect other peroxides. In contrast, the electrochemicaldetection is limited by the absence of a commercialsource and the skill needed to prepare an in-house elec-trode, alignment at microscopic level, and isolate it fromthe high CE potential besides maintaining its daily repro-

ducibility. In our experience, the commercial UV detectoris much more suited for routine analysis than the electro-chemical detector; in addition, it can detect numerouscompounds such as the glucose oxidase enzyme shownin Fig. 3. In other words, these two detectors have differ-ent advantages as well as disadvantages. They serve dif-ferent purposes, but both can be used to monitor enzy-matic activity as demonstrated in Fig. 3, 4.

We checked three different sources, from local drugstores, for their hydrogen peroxide content against theanalytical grade. The result for brand A was 2.98 6 0.14,for brand B 2.94 6 0.08, and for brand C 3.03 6 0.05 g/dL(n = 3). All these concentrations were very close to thelabel on the container (3%).

4 Concluding remarks

This method illustrates that hydrogen peroxide can bemeasured directly without indicator reactions (dyes). Al-though the use of dyes can give higher sensitivity, thesemethods suffer from lack of specificity. The choice of boththe wavelength of 185 nm and concentration on the cap-illary (stacking) greatly improves the sensitivity. Further-more, hydrogen peroxide can be used as a substrate tomonitor the activity of those enzymes involved in its me-tabolism such as glucose oxidase, peroxidase, and cata-lase. The described CE method is simple and rapid. It canbe used directly or on the basis of stacking by ACN.

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