Potentiometric determination of salicylhydroxamic acid (urinary struvite stone inhibitor) based on the inhibition of urease activity

ANALYTICA CHIMICA ACTA ELSEVIER

Analytica

Chimica

Acta 35 1 (1997) 91-96

Potentiometric determination of salicylhydroxamic acid (urinary struvite stone inhibitor) based on the inhibition of urease activity Saad S.M. Hassana,*, Ramadan M. El-Bahnasawyb,

Nashwa M. Rizkb

“Department of Chemistry, Faculty of Science, Ain Shams University, Cairo, Egypt hDepartment of Chemistry, Faculty of Science, El-Menouja University, Menoufia, Egypt Received 21 January

1997; received in revised form 18 April 1997; accepted

11 May 1997

Abstract A novel, sensitive and selective enzymatic potentiometric method for the determination of SHAM drug (salicylhydroxamic acid) is described. It is based on the fast and potent inhibitory action of SHAM on urease-catalyzed urea hydrolysis. The initial rate of the urea/urease reaction is potentiometrically monitored using an ammonia gas sensor. Effects of urease activity, urea substrate concentration, incubation time, temperature and pH are demonstrated. Under optimized conditions, there is a linear relationship between the degree of enzyme inhibition and SHAM concentrations over the range OS-7 pg ml-‘, with a 3u detection limit of 0.1 pg ml-‘. Determination of SHAM in some pharmaceutical preparations shows an average recovery of 98% of nominal and a mean relative standard deviation of 1%. Drug decomposition products and metabolites (salicylic acid and salicylamide) do not interfere. 0 1997 Elsevier Science B.V. Keywords: Enzyme inhibition: Salicylhydroxamic acid (SHAM); Potentiometry; Urea-urease catalytic reaction; Pharmaceutical analysis: Ammonia gas sensor

1. Introduction Struvite or malignant stones are called stone cancer because of their high recurrence rate after surgery and poor prognosis for renal function [l]. These stones are developed in urinary tracts infected by urea-splitting bacteria. The hyperammonuria and alkalinization of urine by bacterial urease are considered to be etiologic factors in the formation of these stones 121. Some hydroxamic acid derivatives such as hydroxybenzhydroxamate (HBHA) or salicylhydroxamic acid

*Corresponding

author. Fax: +20 (2) 83 1836.

0003-2670/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PIZ SOOO3-2670(97)00355-3

(SHAM) are used as therapeutic agent for such a type of urolithiasis [3-51. The potential activity of the drug as trypanocidal [6] and antitubercular agent [7] has also been reported. A few methods are available for determining SHAM including UV-visible spectrophotometry [8-101, and liquid chromatography (LC) [6,1 I]. Whereas the spectrophotometric methods are not sensitive, involve reactions with metal ions under strictly controlled conditions and suffer from interferences by the major drug degradation and metabolite products, the LC methods require several manipulation steps and expensive instrumentation. A direct potentiometric method for determining SHAM using a tin(IV) tetraphenyl porphyrin-based PVC sensor has

92

S.S.M. Hassan et al./Analytica

recently been described [ 121. The sensor allowed the sensitive determination of as low as 5 pg ml-‘, but salicylate interfered significantly. Analytical methods based on enzymatic inhibition have been successfully employed for the determination of some drugs [13], herbicides [14], metal ions [15] and anions [16,17] using potentiometric [13], amperometric [ 141, fluorimetric [ 151 and spectrophotometric [16,17] monitoring techniques. Since the therapeutic action of SHAM and related hydroxamic acid derivatives has been explained on the basis of their ability to act as specific and potent inhibitors of urease [ 181, the present work was undertaken to develop a new potentiometric assay method for the determination of SHAM based on its inhibitory effect on the urease-urea reaction. Under optimized conditions as little as 0.5 ug ml-’ of SHAM was determined with high selectivity and accuracy.

2. Experimental

2.1. Apparatus All potentiometric measurements were made in thermostated 25-ml glass cell at 30fl”C with a Horiba pWmV meter (F-22) and an ammonia gas sensor (Orion 95-12). A nonactin poly(viny1 chloride) (PVC) membrane ammonium ion selective electrode was prepared by a previously described procedure [ 19,201 and used in conjunction with a double junction Ag/AgCl reference electrode (Orion 90-02). The sensor membranes was formulated with 2 wt% nonactin, 69 wt% dibutylsebacate and 29 wt% PVC. Measurement of the initial reaction rate was as previously suggested [20-221. 2.2. Reagents All chemicals used were of reagent grade unless otherwise stated and de-ionised doubly distilled water was used throughout. The enzyme used was jack beans urease (urea: amido hydrolase E.C. 3.5.1.5), ca. 12 000 U g-’ solid supplied by Sigma (St. Louis, MO). Tris(hydroxymethyl)aminomethaneTris, ammonium chloride, hydrochloric acid, sodium hydroxide, PVC powder, dibutylsebacate and tetrahydrofuran were obtained from Aldrich (Milwaukee, WI). Powder

Chimica Acta 351 (1997) 91-96

and pharmaceutical preparations of SHAM (salicylhydroxamic acid) were obtained from El-Nasr Pharm., Egypt. An aqueous 0.1 M solution of Tris buffer adjusted to pH 7.5 with HCl, a stock (2.4x 10e3 M) urea solution, a standard (1 x 10M3 M) SHAM solution and working urease enzymes suspension (240 U ml-‘) were freshly prepared in 0.1 M TrisHCl buffer of pH 7.5, stored in brown bottles and kept in ice. 2.3. Calibration plot for SHAM A 150 ul portion of urease enzyme working suspension{-36 U) was transferred to a 20-ml double jacketed reaction cell thermostated at 30f 1°C containing a small PTFE covered spinbar. A series of aliquots ranging from 20 to 300 ul of lx lob3 M SHAM was added in a separate experiment and the total volume of the solution was made up to 3.0 ml with 0.1 M Tris-HCl buffer of pH 7.5. The solution was stirred for ca. 5 min, the ammonia gas sensor immersed in solution and the potential allowed to reach a constant and stable reading before initiating the reaction. A 3.0 ml portion of urea substrate solution (2.4x lop3 M) was added and the rate curve was recorded. The maximum initial rate of potential change (m V min-‘) was graphically determined by using the rate portion of the curve. The initial reaction rate of urea hydrolysis was plotted as a function of SHAM concentration and the graph was used for subsequent determination of unknown SHAM concentrations. Alternatively, a plot of the % urease enzyme inhibition (Eq. (1)) as a function of SHAM concentration was made. Inhibition

(%) = lOO(A - B)/A,

(1)

where A is the initial rate (mV min-‘) of ammonia liberation obtained in the absence of SHAM, and B is the initial reaction rate (mV min-‘) in the presence of SHAM. 2.4. Determination preparations

of SHAM in pharmaceutical

The content of five tablets or capsules containing SHAM were finally powdered, mixed and weighed. A portion of the powder equivalent to one tablet or capsule was weighed, dissolved in 0.1 M Tris-HCl

S.S.M. Hassan et al./Analytica Chimicn Acta 351 (1997) 91-96

93

buffer of pH 7.5, transferred to a l-l calibrated flask and shaken. The effect of a 60-120 pl portion of the test solution (-3.6 pg ml-’ SHAM in the final reaction solution) on the inhibition of urease was measured as described above and compared with the calibration graph.

3. Results and discussion The degree of inhibition of the urealurease reaction by SHAM was monitored by following the initial rate of urea hydrolysis as a function of SHAM concentration. A potentiometric ammonia gas sensor of the Severinghaus-type and a NH: ion PVC nonactin membrane electrode were used to measure the rate of formation of NH3 gas and NH,f ion, respectively. A series of preliminary investigation was first conducted in order to determine the influence of urease activity, urea substrate concentration, incubation time, temperature and pH on the rates of urea hydrolysis in the absence (control) and presence of SHAM. The difference between the two rates (AE/AT,)dif was taken as a measure of the degree of inhibition caused by SHAM.

Fig. 1. Typical potentiometricresponse curves of the ammonia gas sensor for the hydrolysisof 1.2x 10e3M urea with 6 U ml-’ urease in the absence (A); and presence (B) of 1.5; (C) 3.0 and (D) 4.5 ~g ml-’ SHAM at 30°C and pH 7.5 (Tris-HCIbuffer).

3.1. Effect of w-ease activity Urease is a widely distributed enzyme in flowering plants, bacteria and invertebrates and some enzymatic preparations extracted by some of these sources are commercially available. The cheap and most widely studied and characterized Jack bean urease enzyme was used in this investigation. The effect of urease activity required to catalyze the decomposition of up to 1.2x 10e3 M urea substrate was determined in TrisHCl buffer of pH 7.5 at 30°C. The degree of urea hydrolysis and potential response in the presence and absence of 1S-4.5 pg ml-’ SHAM are shown in Fig. 1. The initial rate of NH3 liberation at pH 7.5 and 30°C in the presence of SHAM and 1.2~ 10e3 M urea substrate in a total volume of 6 ml of the buffer as monitored by the ammonia gas sensor increased with increase of enzyme activity. A significant enzyme inhibition of about 65% was observed with 10 yg ml-’ SHAM and 2 U ml-’ of urease which reaction rate of about displayed an initial 15 mV min-‘. The increase of enzyme activity was

associated with an increase of the initial reaction rate and a decrease in the degree of enzyme inhibition. An activity level of 6 U ml-’ urease was sufficient in the presence of up to 10 pg ml-’ SHAM, to generate enough ammonia with a measurable initial rate (Fig. 2). All subsequent measurements were made with 6 U ml-’ enzyme. 3.2. Effect

qf urea concentration

The rate of hydrolysis of 1.2x10~4-1.2x10-2 M urea in the presence of 3 pg ml-’ SHAM by the action of 6 U ml-’ urease in a Tris-HCl buffer of pH 7.5 at 30°C was also investigated. Two series of experiments were conducted with identical levels of SHAM and urease. The first series involved incubation of SHAM and urease for 5 min until attainment of a steady potential reading followed by addition of urea and measurement of the rate NH=, gas liberation. In the second set urea and SHAM were incubated together

S.S.M. Hassan et al./Analytica

Chimica Acta 351 (1997) 91-96

3.4. EfSect of temperature The effect of temperature on the rate of hydrolysis of 1.2x lop3 M urea with 6 U ml-’ of urease in the absence and presence of 3 pg ml-’ SHAM at pH 7.5 (Tris-HCl buffer) was examined. As the temperature increased, the rate of urea hydrolysis reaction increased up to an optimum of -3O”C, probably due to partial dissociation of some enzyme-inhibitor complex, beyond which rapid enzyme denaturation occurred in the presence or absence of SHAM with a sharp decrease in the rate of reaction. In addition, the potential response of the sensors was not stable above 35°C probably due to baseline drift. All subsequent measurements were made at 30fl”C to obtain maximum reaction rate, reasonable % enzyme inhibition, and stable sensor potential readings. Fig. 2. Degree of inhibition of the hydrolysis of 1.2x 1O-3 M urea in the presence of 10 pg ml-’ SHAM and different urease activities at 30°C and pH 7.5 (Tris-HCL buffer).

and urease was added after establishment of a steady sensor potential. The results of these experiments reveal that: (a) in both cases, 1.2x 1O-3 M of urea was sufficient to oversaturate 6 U ml-’ enzyme; (b) incubation of the enzyme with SHAM was more effective than incubation of urea with SHAM; and (c) a preincubation period of 5 min at 30°C prior to reaction with urea was sufficient to cause significant measurable enzyme inhibition. 3.3. Types of enzyme inhibition The type of urease inhibition with hydroxamic acids has previously been reported as non-competitive [3,23]. The type of inhibition caused by SHAM was also confirmed in the present study where the enzyme inhibition was independent of the urea substrate concentration. The double reciprocal plot of urea substrate and SHAM inhibitor (Lineweaver-Burk plot) gave convergence of the lines at the horizontal intercept similar to that commonly obtained with noncompetitive inhibition [23]. The apparent Michaelis constant (K,) in the presence of SHAM under the present reaction conditions was 1.4 mM. Inhibition by SHAM was reversed by lowering the pH of the test solution to c5 or by dialysis.

3.5. Effect of pH The effect of pH on the inhibition of urease-urea hydrolysis in the presence of SHAM was monitored over the pH range 5-9. The maximum initial rate of urea hydrolysis occurred at pH 6-7, conveniently the same as the pH maximum for substrate activity in the absence of SHAM [23]. Since urease exhibited maximum activity over the pH range 6-7, all kinetic measurements were confined to this pH range. Using the ammonia gas sensor, the reaction was conducted at pH 7.5 to ensure diffusion of enough NH3 gas across the gas permeable membrane. Lower pH values were not suitable because the NH3 sensor is limited to the measurement of NH3 in basic media. It has been reported that even at a pH as low as 7 enough ammonia was produced from urealurease reaction that can be detected with good sensitivity by the NH3 gas sensing electrode [24]. With the ammonium ion PVC nonactin membrane sensor, the reaction was carried out at pH 6.5 (citric-citric acid buffer) as a compromise between optimum enzyme activity and maximum NH: ion concentration to be sensed. However measurements with an NH3 gas sensor offer the advantages of high sensitivity and better detection limit for SHAM. 3.6. Determination

of SHAM

The major electrochemical system based on urea/urease

features of the analytical inhibition by SHAM are

S.S.M. Eassan et al./Analytica Table 1 Analytical

features of the potentiometric

determination

95

Chimica Acta 351 (1997) 91-96

of SHAM using urea/urease

inhibitiona

Parameter

NH3 gas sensor

NH:

nonactin

Temperature (“C) Working buffer (0.1 M Tris-HCl)(pH) Working buffer (0.1 M citratexitric) (pH) Enzyme incubation time (min) Urea concentration (M) Enzyme activity (U ml-‘) Linear range (ug ml-‘) RSD for 3 pg ml-’ Correlation coefficient (r)b Detection limit (pg ml-‘)”

3011 1.5

30fl -

5fl 1.2x10-3 6f0.2 OS-7 31t0.2 0.998 0.1

6.5 5fl 1.2x10-s 6~tO.2 0.5-5 3f0.2 0.993 0.2

PVC membrane

sensor

a Based on the average of 10 measurements. b n=5. ’ 3u value.

given in Table 1. Under the optimized conditions, a linear relationship was obtained between % enzyme inhibition and SHAM concentrations over the range OS-7 ug ml-‘. Each 1.0 pg ml-’ SHAM caused a 10% inhibition of the original enzyme activity. The reproducibility of the method evaluated by the relative standard deviations (RSD) of 10 SHAM samples (2-5 pg ml-’ ) each in triplicate was better than 1%. Least squares regression of the data obtained using the ammonia gas Eq. (2) and ammonium nonactin membrane Eq. (3) monitoring sensors over the linear calibration range gave, respectively, the relationships: Inhibition (1. = 0.998, Inhibition (r = 0.993,

(%‘)=(0.02~0.01)+(10.7fO.l)[SHAM], n = lo),

(2)

(%) =(0.03~0.01)+(14.4f0.1)[SHAM], II = lo),

(3)

where the concentration of SHAM is in ug ml-’ and the slopes and intercepts are expressed with 98% confidence. The 3a detection limit is 0.1 ug ml-’ in each case. These variations may be due to the different working pH range and sensor sensitivity. It has to be pointed out that most of the potentiometric, amperometric, fluorimetric and spectrophotometric assay procedures for anions, cations and organics using enzymatic inhibition reactions covered the determination of only one concentration decade or less of the analyte [13-171. The inhibitory action of some related compounds and metabolites of SHAM such as salicylate and

salicylamide on urease was examined. No significant enzyme inhibition was noticed due to the presence of up to lOO-fold excess of these compounds over urea. This is in a good agreement with the results of other workers that amides (-CONH2) which differ from hydroxamic acids only with regard to the -CONHOH group do not inhibit urease [25]. The effect of some excipients and diluents commonly used in drug formulations (e.g., Tween-80, acacia, glucose, citrate, magnesium stearate) was also tested. These compounds exhibited no effect on the urease activity. The suitability of the proposed assay method for determining SHAM in some pharmaceutical preparations is demonstrated by the data given in Table 2. An average recovery of 98.8% of the nominal with a mean standard deviation of f0.8% are obtained. SHAM in these drugs was also determined by extraction followed by spectrophotometric measurement. The

Table 2 Potentiometric determination of SHAM in some pharmaceutical preparations using the ammonia gas sensor and urea/urease inhibition and direct UV-visible spectrophotometry Drug”

SHAM recovery + S.D.%b Potentiometry

Spectrophotometry

SHAM, capsules SHAM, effervescent

99.110.7 98.4108

98.111.1 96.911.3

tablets

a Products of El-Nasar Pharm., contains 300 mg of SHAM. b Average of five measurements.

Egypt,

each

tablet

or capsule

96

S.S.M. Hassan et al./Analytica

results obtained are also included in Table 2 for comparison. The average recovery was 97.5% of the nominal and the mean standard deviation was 1.2%. Apart from the several manipulation steps involved in this procedure, it does not discriminate among various aromatic carboxylic acids and drug metabolites. The potentiometric urease inhibition method is more selective, gives more precise and accurate results and does not involve any pre-treatment or extraction steps.

4. Conclusions The enzymatic potentiometric determination of SHAM drug based on the inhibition of urease activity, overcomes many limitations imposed by other techniques (e.g., uncertainty of chromogenic reactions, colour stability, low sensitivity, poor selectivity, absence of coloured and turbid matrices, requirements for LC grade solvents and expensive instruments). The proposed method offers the advantages of simplicity, low detection limit (0.1 pg ml-‘), short analysis time (~10 min), high selectivity (in the presence of drug metabolites and decomposition products), high repeatability (>98%) and applicability to coloured and turbid test solutions.

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Chimica Acta 351 (1997) 91-96

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PI A.J. Bamicoat,