Flow injection spectrophotometric determination of isoproterenol using an avocado (Persea americana) crude extract immobilized on controlled-pore silica reactor

Talanta 57 (2002) 135– 143 www.elsevier.com/locate/talanta

Flow injection spectrophotometric determination of isoproterenol using an avocado (Persea americana) crude extract immobilized on controlled-pore silica reactor Karina Omuro Lupetti a, Iolanda Cruz Vieira b, Orlando Fatibello-Filho a,* a

Departamento de Quı´mica, Grupo de Quı´mica Analı´tica, Centro de Cieˆncias Exatas e de Tecnologia, Uni6ersidade Federal de Sa˜o Carlos, Caixa Postal 676, CEP 13.560 -970 Sa˜o Carlos, SP, Brazil b Faculdade de Farma´cia e Bioquı´mica, Uni6ersidade de Cuiaba´ (UNIC), A6. Beira Rio, 3100, CEP 78.015 -480 Cuiaba´, MT, Brazil Received 2 July 2001; received in revised form 29 November 2001; accepted 6 December 2001

Abstract An enzymatic reactor was constructed by the immobilization of polyphenol oxidase (PPO) from avocado (Persea americana) crude extract in an inorganic support of controlled pore silica (CPS), after a previous step of silanization. This inorganic support has been used as an excellent carrier to immobilize this enzyme and the enzymatic reactor was used in a flow injection system for the determination of isoproterenol in pharmaceutical products. The procedure is based on the oxidation reaction of this drug with immobilized PPO and the product obtained was monitored at 492 nm. This system presented an analytical curve from 1.23 × 10 − 4 to 7.38 ×10 − 4 mol l − 1 isoproterenol with a detection limit of 6.25 ×10 − 5 mol l − 1. Recoveries of isoproterenol between 98.5 and 103.1%, a relative standard deviation (R.S.D.) less than 1% (n=10) and 36 determinations per h were obtained. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Isoproterenol; Controlled-pore silica; Flow injection analysis; Reactor

1. Introduction Isoproterenol or isoprenaline (4-[1-hydroxy-2[(1-methylethyl)-amino]ethyl]-1,2-benzenediol) is a b-adrenergic stimulant which has been used for the treatment of primary pulmonary hypertension and also has been employed as an adrenergic acceptor, contributing to the research of other * Corresponding author. Fax: + 55-16-260-8350. E-mail address: [email protected] (O. Fatibello-Filho).

bronchodilators as terbutaline, fenoterol, salbutamol and others [1]. The cardiovascular effects of isoprenaline are compared with the adrenaline and noradrenaline, which can relax almost every kind of the smooth musculature that receives adrenergic nervous, but this effect is pronounced in the musculature of bronchus and also in the gastrointestinal tract. The isoprenaline is better absorbed when dispensed by inhalation. This drug is used to bronchitis, cardiac chock and heart attack.

0039-9140/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 9 - 9 1 4 0 ( 0 1 ) 0 0 6 8 1 - 6

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Nevertheless, the excess of this substance can causes heart failure and arrhythmias [2]. There are very few flow injection procedures cited in the literature for the determination of isoproterenol [3–7]. A flow-injection spectrophotometric procedure for determining adrenaline and isoprenaline based on the reaction of these substances with metaperiodate was described by Nevado et al. [3]. The analytical curve was linear up to 2.0×10 − 4 mol l − l and 120 determinations per h were obtained. Another spectrophotometric flow injection procedure for determining this drug has been developed based on the formation of colored complex between isoproterenol and Fe(II) in aminoacetic–carbonate buffer solution (pH 8.3) and measuring the absorbance at 530 nm [4]. The calibration graph was linear in the isoproterenol concentration range from 4.7× 10 − 5 to 1.4×10 − 3 mol l − 1 with a sample throughput of 40 h − 1. Two flow injection procedures for determination of this drug based on the inhibition of the intensity of chemiluminescence (CL) from the luminol – hypochlorite system was described [5,6]. Finally, a flow procedure using imidazole which catalyzed the decomposition of cathecolamines generating hydrogen peroxide, that was detected by CL using various reagents such as luminol and horseradish peroxidase (HRP), peroxyoxalate, luminol and hexacyanoferrate(III), lucigenin, pyrogallol and purpurogallin was described [7]. Among those reagents, luminol CL reaction catalyzed by HRP was recommended for the detection of peroxide in this procedure for isoproterenol. Nevertheless, any enzymatic procedure for determining this analyte was described in the literature. Inorganic support materials have been shown to be excellent carriers to immobilized enzymes. From the analytical point of view, covalent binding of an enzyme to a support is probably the most interesting method of immobilization. Thus, it must be carried out in chemical conditions that are compatible with the stability properties of the proteins. Usually, the immobilization involves two steps: (a) activation of the support and (b) attachment of the enzyme to this support. The characteristics of the inorganic support material are extremely important and are definitives in the

choice of the proper carrier to the enzyme to be immobilized [8–10]. One of the most important factors in choosing the carrier is the pore diameter relative to surface area. There will always be an optimal pore diameter for an enzyme. The relationship between pore diameter and surface area is inversely proportional. The choice of particle size also has a major impact on the activity of an immobilized enzyme. The larger the particle, the greater the effect of diffusion control. Generally, the smallest particle size is the best choice for enzyme immobilization, nevertheless it must be considered the increasing of hydrodynamic resistance with the decreases of particle size, specially in systems involving flow injection analysis [10,11]. There is an increasing interest of the application of crude extracts and/or plants instead of purified enzymes in the construction of biosensors and/or enzyme reactors [12–15]. The use of such materials offers frequently some advantages over those procedures that use pure and isolated enzymes, mainly high content of enzyme, low cost and better lifetime [15]. In this article, a simple, sensitive and rapid flow injection system for determining isoproterenol in pharmaceutical products is reported. A crude extract of avocado (Persea americana) was used as the enzymatic source of polyphenol oxidase (PPO; EC 1.14.18.1) in the construction of controlledpore silica (CPS) reactor. The procedure is based on the oxidation reaction of this drug with immobilized PPO. Thus, this enzyme catalyses the ortohydroxylation of isoproterenol to isoproteroquinone. This quinone is converted to leucoisoproterochrome and oxidized to isoproterochrome, which presents a strong absorption at 492 nm.

2. Experimental

2.1. Apparatus A DuPont Instruments (Newtown, CT, USA) Model RC-5B centrifuge, provided with a Model SS-34 rotor, was used in the preparation of avocado crude extract. A Hewlett–Packard (Boise,

K.O. Lupetti et al. / Talanta 57 (2002) 135–143

ID, USA) Model 8452A UV– visible spectrophotometer with a quartz cell (optical path of 1 cm) was used in PPO activity measurement and total protein determination. An eight-channel Ismatec (Zurich, Switzerland) Model 7618-40 peristaltic pump supplied with Tygon pump tubing was used for the propulsion of the fluids. The manifold was constructed with polyethylene tubing (0.8 mm i.d.). Sample injection was performed using a laboratory-constructed three-piece manual commutator [12] made of Perspex, with two fixed side bars and a sliding central bar, that is moved for sampling and injection. Flow injection spectrophotometric measurements were carried out using a Femto spectrophotometer (Sa˜ o Paulo, Brazil) Model 435 with a glass flow cell (optical path 1.0 cm) connected to a two-channel strip-chart recorder Cole Parmer (Niles, IL, USA) Model 12020000. The effect of temperature on the enzymatic reaction was evaluated using a Tecnal (Piracicaba, Brazil) Model TE 184 thermostatically controlled waterbath.

2.2. Reagents and solutions All reagents were of analytical-reagent grade and all solutions were prepared with water from a Millipore (Bedford, MA, USA) Milli-Q system (Model UV Plus Ultra-Low Organics Water). Isoproterenol was purchased from Sigma (St. Louis, MO, USA) and a 2.5 ×10 − 2 mol l − 1 stock solution was prepared daily in 0.1 mol l − 1 phosphate buffer solution (pH 7.0). The reference isoproterenol solutions were prepared by appropriate dilutions of the stock solution in the same buffer. The Polyclar Super R used as a protective and/or stabilizer agent in the crude extract preparation was kindly donated by GAF (Wayne, NJ, USA). Healthy avocado (P. americana), purchased from a local producer was used as a source of PPO (E.C.1.14.18.1). CPS utilized as inorganic support, was kindly donated by Professor Dr Trevisan from Department of Technology and Application of Institute of Chemistry of Araraquara/SP (UNESP). In this work the following particle diameters in the range

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of 75 –106; 106–150; 150–250; 250– 350 and 355– 500 mm and pore diameter of 173 A, were investigated as inorganic support. Solutions of 2% v/v (3-aminopropyl)triethoxysilane and 2.5% w/w glutaraldehyde purchased from Sigma were utilized for silanization and activation of the support of CPS, respectively. Three Brazilian samples containing isoproterenol were analyzed using the proposed flow injection procedure.

2.3. A6ocado crude extract preparation A mass of 25 g of the fresh peeled avocado was homogenized in a blender with 100 ml of 0.1 mol l − 1 phosphate buffer (pH 7.0) containing 2.5 g of Polyclar Super R for 2 min at 4–6 °C. The homogenate was rapidly filtered through four layers of cheesecloth and centrifuged at 15 000 rpm for 20 min at 4 °C. The resulting supernatant was stored at this temperature in a refrigerator and used as the enzymatic source after the determination of the PPO activity and total protein [12].

2.4. PPO acti6ity and total protein determinations PPO activity present in the crude extract was determined in triplicate by measurement of the absorbance at 420 nm of p-quinone produced by the reaction between 0.2 ml of supernatant solution and 2.8 ml of 0.05 mol l − 1 catechol solution in 0.1 mol l − 1 phosphate buffer (pH 7.0) at 25 °C. The initial rate of catechol reaction was a linear function of time for 1.5–2.0 min. One activity unit is defined as the amount of enzyme that causes an increase of 0.001 absorbance units per min under the experimental conditions described above [12–14]. Total protein concentration was determined in triplicate using bovine serum albumin as standard as described by Lowry et al. [16].

2.5. Immobilization procedure Silica is one of the best support employed to immobilize enzymes, because it is inert to the microbial attacks, is stable and also easy han-

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dling. The beads were initially prepared by silanization of silica by (3-aminopropyl)triethoxysilane, then the activation of support with glutaraldehyde and finally covalent attached of glutaraldehyde to active enzymes [10].

2.6. Silanization of CPS To a mass of 1.0 g of clean silica was added 3 ml of 2% v/v (3-aminopropyl)triethoxysilane aqueous solution. Then, this inorganic support was coated with the (aminoalkyl)silane for 30 min in a vacuum desiccator. After removing the excess of solution, the inorganic support was heated by 12– 15 h at 70–80 °C. The silanized silica was washed with distilled water, dried in a 100– 110 °C oven for at least 8 h and the product was stored for later use.

2.7. Acti6ation of inorganic support with glutaraldehyde Initially, a mass of 0.5 g of silanized silica was transferred to a vacuum tube, 1.0 ml of a 2.5% v/v glutaraldehyde solution was added and let to react under suction of a water vacuum pump. Appearance of a red color indicates that the glutaraldehyde was attached to the silane. After a 30 min reaction time, the particles of the inorganic support were washed with distilled water until remove the excess of glutaraldehyde and finally washed with 0.1 mol l − 1 phosphate buffer solution (pH 7.0).

experiment it was possible to determine the concentration of enzyme immobilized on the CPS (units of PPO per g of CPS). After that, the inorganic support containing immobilized PPO was washed with a 0.1 mol l − 1 phosphate buffer solution (pH 7.0), until the excess of enzyme has been removed. The enzyme immobilized in the support was then stored in a 0.1 mol l − 1 phosphate buffer solution (pH 7.0), at 4 °C in a refrigerator and used when necessary in the confection of the enzymatic reactors.

2.9. Preparation of enzymatic reactor and flow-injection procedure A mass of 0.2 g of silica with immobilized enzyme was packed in an glass tube (3.0 mm i.d. and 30 mm long) containing small pieces of glass wool in the extremities to avoid the leakage of this material. This reactor was used in a one-channel flow injection system with spectrophotometric flow cell as showed in Fig. 1.

3. Results and discussion

3.1. Immobilization of the PPO of a6ocado crude extract on the CPS The immobilization of PPO by covalent attachment to CPS supports involves reactions that are similar to the covalent attachment of enzymes to

2.8. Co6alent attachment of the PPO in the acti6ated inorganic support A 5.0 ml aliquot of avocado crude extract solution containing 5700 units of PPO per mg of protein was transferred to 0.5 g of the activated inorganic support and let to react by 48 h at 4 °C. This coupling reaction was monitored by determination of enzymatic activity of supernatant at the following times: 4, 8, 16, 24 and 48 h. Simultaneously a control experiment was carried out using 0.5 g of CPS without activation at the same experimental conditions. So with the difference between the enzymatic activities obtained in each

Fig. 1. Schematic diagram of the flow injection system used for the spectrophotometric determination of isoproterenol. The central bar of the manual injector – commutator (I) shows the injection position after commutation. P, peristaltic pump; S, sample or reference solutions; L, sample loop (375 ml); C, carrier solution (0.1 mol l − 1 phosphate buffer solution at flow rate of 2.1 ml min − 1); R, enzymatic reactor (3.0 mm i.d. and 30 mm long); D, spectrophotometric detector at 492 nm and W, waste, at 25 °C.

K.O. Lupetti et al. / Talanta 57 (2002) 135–143

Fig. 2. Enzymatic activity (units of PPO immobilized per gram of CPS) obtained for particle diameter of 106 –150 mm and pore diameter of 173 A, as a function of time (h).

organic supports [17,18]. As discussed before, CPS has been shown to be an excellent carrier to immobilized enzymes and involves two steps such as activation of the support material and attachment of PPO to this support. Considering that the characteristics of CPS play an important role in the enzyme immobilization, the effect of particle diameter on the PPO immobilization was initially investigated. Following the procedure described above, in this work particle diameters in the range of 75 –106; 106–150; 150–250; 250– 350 and 355– 500 mm and pore diameter of 173 A, were used as inorganic support. Monitoring the PPO activity of supernatant in the 0.1 mol l − 1 phosphate buffer solution (pH 7) by 48 h, it was observed that the reaction of immobilization of PPO in CPS was completed after 24 h for these particles. Among these particle diameters investigated, the particle diameter of 106–150 mm presented the best enzyme loading analytical signal. Fig. 2 presents the enzymatic activity (units of PPO immobilized per gram of CPS) obtained for particle diameter of 106–150 mm as can be seen from this figure, after 24 h of immobilization time, 2280 units of PPO per gram of CPS were immobilized.

3.2. Flow-injection system and reaction conditions Preliminary studies were carried out using the manifold that is shown in Fig. 1 to establish the

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optimal enzymatic reaction conditions between isoproterenol and PPO immobilized on the inorganic support. The effect of flow injection parameters, pH of carrier solution and temperature were initially evaluated. The best analytical signal (S/N) was obtained for a flow rate of 2.1 ml min − 1, sample loop of 375 ml, enzymatic reactor of 3.0 mm i.d. and 30 mm long and 0.1 mol l − 1 phosphate buffer solution at pH 6.0. The same optimum pH value for PPO activity was found by Vieira and Fatibello-Filho [12,13] in pure and crude extracts of sweet potato (Ipomoea batatas (L.) Lam). Thus, when the sample zone containing isoproterenol contact the PPO immobilized on CPS in the reactor, this enzyme catalyses the ortho-hydroxylation of isoproterenol to isoproteroquinone. This quinone is converted to leucoisoproterochrome and oxidized to isoproterochrome which presents a strong absorption at 492 nm [19,20] (Fig. 3). The tubular coiled reactor was placed in a water-bath and the effect of temperature was investigated between 25 and 50 °C. The PPO exhibited the highest activity in the temperature range of 25–35 °C after which a gradual decline in its activity by heat inactivation was observed between 35 and 50 °C. Considering the high stability of PPO at 25 °C, this temperature was selected for further experiments. Table 1 summarizes all results obtained in this flow injection parameters, pH and temperature studies.

3.3. Reco6ery, repeatability, reproducibility, interference and lifetime studies Recoveries of 98.5–103.1 of isoproterenol from three commercial products (n= 5) were obtained using this flow injection procedure with the enzymatic reactor. In this study, 1.5× 10 − 4, 3.0× 10 − 4 and 4.5×10 − 4 mol l − 1 of isoproterenol solutions were added to each sample and the analytical signal (absorbance) was obtained. The recovery results obtained suggested an absence of matrix effect on those determinations. The relative standard deviation was less than 1% for ten injections of a solution containing 2.0× 10 − 4 mol l − 1 isoproterenol in 0.1 mol l − 1 phosphate buffer solution (pH 6.0).

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Fig. 3. Reaction steps of the catalytic oxidation of isoproterenol by PPO of avocado crude extract immobilized on the CPS.

The reproducibility of five enzyme reactors shows only a slight variation of ca. 3% of analytical curve slope. The effect of excipient substances frequently found with isoproterenol in pharmaceutical formulations, such as lactose, sucrose, fructose, starch, poly(ethylene glycol), sodium chlorine and magnesium stearate were evaluated using the proposed flow procedure. The ratios of the concentrations of isoproterenol to the excipient substances were fixed at 0.1, 1.0 and 10.0. None of these substances interfered in the developed enzymatic method. After a 7–8 h working period, no baseline drift was observed and only a slight variation of 3 – 4% of response slope was obtained. One enzymatic reactor was used during 15 days and the sensitivity of flow injection procedure decreased to 80% of its initial value and at least 400 assays could be performed with a single reactor, confirming, as expected, the high stability of avocado (P. americana) crude extract immobilized on CPS. This reactor when not in use was stored at 5 °C in a refrigerator. In addition, maintaining the reactor in this experimental condition, the enzymatic activity was kept constant by at least 7 months.

3.4. Analytical characteristics and application A series of reference and sample solutions of

isoproterenol were inserted in triplicate into the manifold presented in the Fig. 1 under the selected conditions. Triplicate signals for six (1.23; 2.46; 3.69; 4.92; 6.15 and 7.38× 10 − 4 mol l − 1) isoproterenol reference solutions (Abs=9.8× 10 − 3 + 3.3× 102C; r=0.9985, where Abs is the absorbance and C the concentration of isoproterenol in mol l − 1), triplicate signals for three pharmaceutical formulations (A, B and C) and reference solutions again showed good precision and baseline stability as can be seen in Fig. 4. The analytical curve for isoproterenol was linear in the concentration range from 1.23× 10 − 4 to 7.38× 10 − 4 mol l − 1 with a detection limit of 6.25×10 − 5 mol l − 1 (three-fold blank standard deviation/slope) and 36 determinations per h were obtained. A comparison between the proposed flow injection method and that one developed by Nevado et al. [3] shows that the former preTable 1 Optimization of FI parameters FI parameter

Range studied

Optimal value

Injected volume (ml) Reactor (cm) Flow rate (mL min−1) pH Temperature (°C)

25–750 1.0–5.0 1.2–2.8 5.0–7.5 25–50

375 3.0 2.1 6.0 25

K.O. Lupetti et al. / Talanta 57 (2002) 135–143 Fig. 4. Transient absorbance signals obtained in triplicate for reference isoproterenol solutions (1.23; 2.46; 3.69; 4.92; 6.15 and 7.38 × 10 − 4 mol l − 1), three pharmaceutical products (A, B and C) and the reference solutions again, at 25 °C. Also, the analytical curve obtained is shown inset this figure.

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Table 2 Determination of isoproterenol in pharmaceutical products using the pharmacopoeial [22] and flow injection procedures Sample

A B C

Isoproterenol (mol l−1)

Relative error (%)

Label

Pharmacopoeial

FIA

E1

E2

1.25×10−4 2.50×10−4 3.75×10−4

1.30×10−4 9 0.03 2.55×10−4 9 0.02 3.72×10−4 9 0.04

1.26×10−4 90.02 2.52×10−4 90.03 3.70×10−4 90.05

+0.8 +0.8 −1.3

−3.0 −1.2 −0.5

n= 5, Confidence level 95%; E1 = FIA vs. label; E2 = FIA vs. pharmacopoeial.

sented a narrow concentration linearity and higher detection limit. Nevertheless, the isoproterenol concentration in pharmaceuticals is high and can be successfully determined by the proposed procedure. In addition, the presented flow injection manifold is simpler than that one described in the literature [3] with minimum toxic effluent generation, in accord to the strategies to greener analytical chemistry [21]. The proposed flow injection procedure was validated by applying it to the determination of isoproterenol in pharmaceutical products. Table 2 presents the results obtained for three commercial samples using a pharmacopoeias [22] and the proposed flow injection procedure. Applying a paired t-test to the results obtained by either procedures, it was found that all results are in agreement at 95% confidence level and within an acceptable range of error.

4. Conclusions The flow injection procedure using an avocado (P. americana) crude extract immobilized on CPS reactor reported in this paper is reliable, simple, rapid, low cost, precise and does not require extensive preliminary sample treatment.

Acknowledgements Financial support from FAPESP (Processes 1991/2637-5 and 1994/4822-2) PADCT/CNPq

(Process 62.0060/91-3), CNPq (Process 50.1638/ 91-1), scholarship granted by FAPESP (Process 98.512-2) to Karina Omuro Lupetti are gratefully acknowledged. Also we would like to thank Professor Dr Trevisan for the donation of CPS.

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