Determination of ethamsylate in pharmaceutical preparations by irreversible biamperometry

Microchemical Journal 80 (2005) 65 – 70 www.elsevier.com/locate/microc

Determination of ethamsylate in pharmaceutical preparations by irreversible biamperometry Jia-Quan Chen, Jun-Feng Song*, Liang-Feng Chen Institute of Analytical Science, Northwest University, Xi’an 710069, PR China Received 16 June 2004; received in revised form 2 November 2004; accepted 5 November 2004 Available online 12 January 2005

Abstract A novel flow-injection irreversible biamperometric method is described for the determination of ethamsylate. The proposed method is based on the oxidation of ethamsylate at one platinum electrode and the reduction of permanganate at another to form an irreversible biamperometric detection system. Ethamsylate can be determined over the range 1.010 6–1.010 4 mol l 1 with a sample measurement frequency of 180 samples h 1. The detection limit for ethamsylate is 4.010 7 mol l 1. The stability of the proposed method is shown by a RSD of 0.52% for 11 replicate determinations of 2.010 5 mol l 1 ethamsylate. The proposed method was applied to the determination of ethamsylate in pharmaceutical preparations. D 2004 Elsevier B.V. All rights reserved. Keywords: Ethamsylate; Flow injection; Irreversible biamperometry; Permanganate

1. Introduction The attractive potential of electrochemical detector in flow-injection analysis (FIA) has been shown in many areas of analytical chemistry. Among various electrochemical detectors, biamperometric detector [1–4] with two identical platinum electrodes polarized with a small potential difference has some special advantages. The instrumentation and the flow through cell are simple and the detection conditions are easy to control. Moreover, based on the principle of dead-stop end point detection, the method shows high sensitivity, high selectivity and signal to noise (S/N) ratio owing to a small potential differences applied (usually b200 mV). Unfortunately, the biamperometry is only suitable to few reversible couples such as I2/I and Br2/Br systems. For analytes with irreversible oxidation or reduction, two detection schemes have been employed. One scheme is the conventional amperometry. In this scheme, a larger constant-controlled potential even much larger has to be applied to obtain current signal, which will inevitably lead to the * Corresponding author. Fax: +86 29 88303448. E-mail address: [email protected] (J.-F. Song). 0026-265X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.microc.2004.11.002

decrease of selectivity and S/N ratio. The other is the socalled indirect biamperometry based on homogeneous reaction of the analyte with a reduced or oxidized form of the indicating reversible couple to produce the other oxidized or reduced form needed, the selectivity of this scheme is dependent on the specificity of the very limited homogeneous reactions [1]. In view of the limitations of two schemes mentioned above, therefore, it is needed to establish a more simple and available detection scheme for the determination of analyte with irreversible character in electrode process. In our previous work, the flow-injection biamperometry for irreversible couple was introduced [5,6]. In the scheme, the biamperometric detection was established by coupling two irreversible and independent couples that their electrode processes were inverse and half-wave potentials E 1/2 (or peak potential E p) were close to each other as possible, resulting to the existence of a couple-combined system similar to the reversible couple system. The resulting current was limited by the smaller one between anodic and cathodic currents that were related to the concentration of each reactant respectively. When the concentration of one reactant was high enough, the linearity between the cell

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current and the concentration of the other reactant of interest could be obtained over certain range. The method was named as irreversible biamperometry. According to this scheme, any analyte, either being oxidized or reduced, even if it was of high redox potential, can be determined so long as the analyte was coupled with another selected substance that possesses inverse electrode process and half-wave potentials E 1/2 (or peak potential E p) closed to that of the analyte. The method not only inherited the inherent advantages of biamperometry for reversible couples but also largely extended the application scope of biamperometry. Additionally, it allowed carry out the determination with a very small applied potential difference DE even 0 V. Since the applied DE was very small, the method showed high selectivity and S/N ratio, and had been successfully applied to detect hydroxylamine [7], cysteine [8] and hydrazine [9] by coupling the reduction of PtO at one platinum wire electrode and the oxidation of these analytes with low oxidation potential at another one. Ethamsylate (2,5-dihydroxybenzenesulfonic acid with diethylamine) is a systemic, nonthrombogenic haemostatic agent that has been used for the prophylaxis and control of hemorrhages due to the rapture of small blood vessels in surgery and in clinical conditions. Few works had been published for the determination of ethamsylate. Potentiometry [10], spectrophotometry [11] and chemiluminescence [12–15] methods were proposed, but in some cases, those methods required carefully to control measurement conditions or suffered from low selectivity. Therefore, the determination of ethamsylate requires a simple, quick and sensitive method. However, FIA with electrochemical detector for the determination of ethamsylate has not been found in literature. In present work, a new irreversible biamperometric method for the determination of ethamsylate was proposed by using two identical pretreated platinum electrodes with an applied potential difference DE of 0 V. As ethamsylate was found to be irreversibly oxidized at the platinum electrode at much high positive potential, the biamperometric detection scheme was established by coupling it with an irreversible reduction of permanganate owing to the reduction potential of permanganate near to the oxidation one of ethamsylate at two platinum electrodes, respectively. With the advantages of the applied DE of 0 V, satisfactory sensitivity, selectivity and high S/N were obtained. The proposed method was suitable for automatic and continuous analysis and was tested for determination of ethamsylate in pharmaceutical preparations.

(National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China) in 100 ml water. The stock solution was kept in an opaque brown plastic bottle and stored in the refrigerator. The standard working solutions were prepared daily from the stock solution by appropriate dilution with water. Potassium permanganate solution (1.010 3 mol l 1) was prepared by dissolving 0.0158 g potassium permanganate (Xi’an Chemical Plant, Xi’an, China) in 100 ml of 0.05 mol l 1 H2SO4 solution. All reagents used were of analytical grade unless specified otherwise. Twice distilled water was used throughout the experiments. 2.2. Instrument A CHI660 electrochemical workstation (CH Instruments, USA) equipped with a personal computer was used to impose the potential difference and to record the resulting current. Additionally, it was used to perform the cyclic voltammeter experiments. The schematic diagram of the homemade biamperometric detector, which was constructed from a Teflon rod, was shown as Fig. 1. The two electrode rooms in the detector were separated by means of a salt bridge. The internal volume of each room was estimated to be 20 Al (1.2 cm length, 0.7 mm i.d.). The platinum wire electrodes (1.1 cm length, A 0.5 mm) were pretreated electrochemically by alternating polarization between +1.55 and 0.2 V in 0.05 mol l 1 H2SO4 solution after soaked with concentrated nitric acid for 5 min and rinsed with water prior to every measurement. A potential difference DE was imposed between two platinum wire electrodes of the biamperometric detector by connecting both the auxiliary electrode and the reference electrode led to one side of the detector and the working electrode led to the other side. A dual carrier manifold was constructed with polyethylene tubing (0.8 mm i.d.) to perform the detection. The system included a model IFIS-B automatic sampling system (Ruike Electronic Instrument Limited, Xi’an, China), which consisted of two peristaltic pumps and a six-way injection valve, controlled by a microcomputer. The cyclic voltammograms were performed independently with a three-electrode system, namely, a small-sized PC

S CHI600

P

T D

2. Experimental

B

A

C E

E F

2.1. Reagents and material 2

Standard stock solution of ethamsylate (1.010 mol l 1) was prepared by dissolving 0.2633 g of ethamsylate

Fig. 1. Schematic diagram of the flow-injection biamperometric detection system. (P) peristaltic pump, (V) valve, (S) sample, (C) carrier solution, (D) biamperometric detector, (A) sample inlet, (F) sample outlet, (E) platinum wire electrode, (B) KMnO4 solution inlet, (T) salt bridge.

J.-Q. Chen et al. / Microchemical Journal 80 (2005) 65–70

67

platinum wire working electrode, a platinum wire auxiliary electrode and a saturated calomel electrode (SCE). Unless otherwise mentioned, potentials in present work were referred to the potential of the saturated calomel electrode. An AIC UV-900 spectrophotometer (Beijing Ruili Instrument, China) with a cell of 1 cm path length and a wavelength of 305 nm was used for measuring the absorbances of ethamsylate versus the black solution.

irreversible oxidation wave P2 appeared at about 0.83 V with the wave P1 keeping changeless (Fig. 2(a)). The wave P2 was due to two-electron oxidation of ethamsylate [17].

2.3. Procedure

As shown in Fig. 2(a), the potential difference between the oxidation potential of ethamsylate and the reduction potential of platinum oxide was about 0.63 V. If a biamperometric detection system was established by using these two couples, a large potential difference DE imposed between two electrodes would have to be applied to obtain current response. Undoubtedly, the increasing of the applied DE will lead to the decrease of selectivity and S/N level. In order to obtain current response when the applied DE was very smaller even zero, another irreversible couple with opposite electrode process and close E 1/2 (or E p) to that of ethamsylate was needed. Among various alternatives, permanganate was a good choice. Fig. 2(b) showed that adding 1.010 3 mol l 1 KMnO4 led to two reduction waves P3 and P4. The wave P3 was the same as the wave P1, the reduction of platinum oxide, and the wave P4 at about 0.79 V corresponded to the reduction of MnO4 to Mn2+, respectively. Fig. 2 showed that the oxidation wave P2 and the reduction wave P4 were separately in anodic and cathodic polarized curves; they were from two independent and irreversible couples with the peak potential difference of only 0.04 V. According to the irreversible biamperometry, the biamperometric detection scheme was established by coupling the oxidation of ethamsylate with the reduction of permanganate. In this case, the determination can be carried out with a small applied DE even 0 V. In addition, since a homogeneous redox reaction of ethamsylate with MnO4 could occur if they were mixed together, they were separated into two electrode rooms by means of a salt bridge in the detector. Therefore, the influences from the homogeneous reaction were avoided.

A potential difference DE of 0 V was kept across the two platinum wire electrodes and the cell current was recorded by the CHI660 workstation. By keeping the valve in the sampling position, 0.5 mol l 1 H2SO4 carrier solution at the rate of 5.6 ml min 1 was continuously pumped into one electrode room of the biamperometric detector and 20 Al of potassium permanganate solution was sealed in the other electrode room and keeping immobile. When baseline was established on the recorder, 150 Al of standard or sample solution was injected into the carrier solution. Calibration graphs of the cell current versus ethamsylate concentration were plotted and the content of each sample was determined.

3. Results and discussion 3.1. Cyclic voltammetric studies Voltammetric behaviors of ethamsylate and MnO4 at platinum wire electrode were examined in the potential range from 0.2 to 1.55 V, respectively (Fig. 2). In 0.5 mol l 1 H2SO4 solution, a reduction wave P1 appeared at about 0.20 V, which had been extensively studied and attributed to the reduction of platinum oxide [16]. After adding a certain amount of ethamsylate in 0.5 mol l 1 H2SO4 solution, a new

1.5

p1

Current /1e-4A

1.0

a

p4

p3

b

0.5

b

-1.0 -1.5 -2.0 1.4

SO3H O

OH NH(C2H5)2 HO

NH(C2H5)2 + 2H+ + 2e– O

3.2. Effect of the applied potential difference

0.0 -0.5

SO3H

p2

a

1.2

1.0

0.8

0.6

0.4

0.2

0.0

Potential /V (vs, SCE) Fig. 2. Cyclic voltammograms of ethamsylate/permanganate biamperometric system. (a) The presence of 1.010 3 mol l 1 ethamsylate in 0.5 mol l 1 H 2 SO4 solution and (b) the presence of 1.010 3 mol l 1 permanganate in 0.05 mol l 1 H2SO4 solution. Initial potential 0 V, reversal potential 1.4 V, scan rate v=100 mV s 1.

In irreversible biamperometry, the potential difference DE imposed between two electrodes dependents on the DE 1/2 (or DE p) of the half-wave potential E 1/2 (or peak potential E p) of two irreversible couples. Generally, when the DE 1/2 (or DE p) is large, the increasing of the applied DE leads to the increase of sensitivity. In present work, when the applied DE was 0 V, the true potentials of two electrodes were between 0.83 V and 0.79 V and were kept the same value as anodic and cathodic currents were equivalent to each other. Under the under-potential condition, the current between the two electrodes did not reach the limiting current values of both reactants,

J.-Q. Chen et al. / Microchemical Journal 80 (2005) 65–70

increasing the applied DE will make the potentials of two electrodes move to the limiting current direction of one or the other reactant, and will lead to the increase of sensitivity. However, the increase of the applied DE will require the usage of an additional potential-controlled setup and will accompany the decrease of both selectivity and S/N ratio, which was not expected in practical determinations. Fig. 3 showed the influence of the applied DE on the S/N ratio. When the applied DE was 0 V, the obtained S/N ratio was the maximum value. Therefore, the applied DE of 0 V was chosen in present work.

10 9 8

b

7

Current /1e-6A

68

6 5 4 3

a 2 1 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5

3.3. Selection of carrier solution

pH

The carrier solution was selected based on the effect of pH value of carrier solution on both peak height and peak width of the current response. The effect of pH value of carrier solution was investigated in such carrier solution as H2SO4 solution in various concentrations and BrittonRobinson buffer of different pH values over the range 1.81 to 7.96, with the applied DE of 0 V. Since ethamsylate is rapidly oxidized by dissolved oxygen in alkaline medium, acidic solution, therefore, is preferable. As shown in Fig. 4(a), the cell current decreased with the increase of pH value of the carrier solution, the carrier solution with low pH value was, therefore, favorable to obtain high sensitivity. Therefore, three strong acid solutions, H2SO4, HNO3 and HCl, were examined. The detector showed more stable response and well-defined peak shape in H2SO4 solution than that in HCl and HNO3 solutions. The current signal increased with the increase of H2SO4 concentration (Fig. 4(b)), accompanied with unstable baseline and distortion of peak shape. Moreover, using H2SO4 solution was to avoid the corrosion of the working platinum electrode by HNO3 and HCl solutions in one electrode room and to avoid the reaction of HCl with permanganate in another one. So there was a tradeoff; in present work, 0.5 mol l 1 of H2SO4 solution was selected as the carrier solution.

3.4. Selection of KMnO4 concentration To obtain good linearity between current response and ethamsylate concentration, KMnO4 concentration should be kept enough high, because the current flowing through the detector was limited by the smaller one between the anodic current of ethamsylate and the cathodic current of MnO4 . 1.010 3 mol l 1 KMnO4 solution was selected because ethamsylate concentration in present work was generally less than 1.010 4 mol l 1. In virtue of that, KMnO4 concentration was much higher than that of ethamsylate; the consumption of KMnO4 reduction could be negligent. Even when 20 Al of 1.010 3 mol l 1 KMnO4 solution was sealed in one electrode room and keeping immobile throughout the experiments, the obtained current response was not remarkably different compared with a large volume of 1.010 3 mol l 1 KMnO4 solution flowing in the electrode room. In addition, no obvious influence was observed when using 20 Al of 1.010 3 mol l 1 KMnO4 solution for no less than 200 samplings. So a 20 Al of 1.010 3 mol l 1 KMnO4 solution was chosen. Certainly, it is necessary that the permanganate solution is often renewed.

60

3.5. Optimization of FIA system

50

Since no homogeneous reaction occurred in the proposed method, considerations for some conditions like reaction time and temperature were needless. Such parameters of the FIA operation as flow rate, injection volume and others were optimized by using 1.010 5 mol l 1 ethamsylate standard solution. Results showed that both peak height and peak width of current response increased with increasing injection volume. When the injection volume exceeded 150 Al, the peak height reached a plateau. The choice of the flow rate must also take into account the changes in peak height and peak width as well as the rate of the return to the baseline, which influenced the sampling throughput. In present work, the peak height

40

S/N

Fig. 4. Effect of pH value on cell current in (a) Britton-Robinson buffer, (b) H2SO4 solution. The applied DE=0 V, flow rate r=5.6 ml min 1.

30 20 10 0 -0.4

-0.3

-0.2

-0.1

0.0

0.1

0.2

Potential difference/ V Fig. 3. Effect of potential difference applied on signal/noise (S/N) ratio. C=2.010 5 mol l 1 ethamsylate, flow rate r=5.6 ml min 1.

J.-Q. Chen et al. / Microchemical Journal 80 (2005) 65–70 50

3.7. Interference study

50

10

40

40

8

-7

i /10 A

30

6

20

4

-7

i /10 A

30

10

20

0 0

10

2

4

6

-5

8

-1

10

12

2

C /10 mol l 1 0.8 0.6 0.4 0.1 0.2

0 0

69

100

200

300

400

500

600

700

800

Time /s

Fig. 5. Current responses to ethamsylate standard solutions (concentrations on the peaks refer to 10 5 mol l 1 ethamsylate). The applied DE=0 V, flow rate r=5.6 ml min 1.

was found decreased with the flow rate increasing from 0.5 to 6 ml min 1. At low flow rate, however, the peak broadened. Thus, compromises were needed. In consideration of these factors, the injection volume and the flow rate were chosen as 150 Al and 5.6 ml min 1, respectively. The distance between the detector and the valve had no obvious effect on current response and the distance was 24 cm. 3.6. Stability of the method Generally speaking, the anodic oxidation of aromatic compounds at solid electrodes led to the loss of electrode response and precision due to the formation of polymeric films on electrode surfaces. However, no loss of electrode response was observed for the detection of aromatic compounds in low concentration [18]. Since the ethamsylate concentration in present work was less than 10 4 mol l 1, the sampling rate was fast enough to eliminate the polymerization of the oxidation product of ethamsylate and the platinum wire electrodes were cleaned electrochemically prior to each measurement; the contamination of the electrode was not found in the proposed FIA biamperometric determinations. Under the selected conditions, the detector showed long-term stability even at low flow rate. The RSD observed for 27 sequential determinations of 1.010 5 mol l 1 ethamsylate was 3.1%. As many as 400 times repetitive injections were performed and the results showed good durability of the detector.

The effects of various inorganic ions and organic compounds commonly existed in pharmaceuticals on the determination of 1.010 5 mol l 1 ethamsylate were studied. The tolerance limit is defined as the molar ratio of additive to ethamsylate causing less than F5% relative error. Since the applied DE was 0 V, the detector was free from interference induced by oxidation or reduction of the additives. The tolerance limits of additives to 1.010 5 mol l 1 ethamsylate were z500-fold amount of Ag+, Ni2+, Al3+, Mn2+, Zn2+, Co2+, Cu2+, Bi3+, Mg2+, Cd2+, La3+, Na+, K+, SO42 , CO32 , PO43 , Cl , NO3 , 100-fold amount of glucose, lactose, starch, sucrose, mannit, epthedrina, lvaline, l-threonine, l-cysteine, l-serine, l-glutamic acid, larginine, l-histidine and 10-fold amount of uric acid, vitamin C, respectively. 3.8. Calibration curve, detection limit and precision Under the optimized conditions, the biamperometric detector used had good response to ethamsylate standard solutions (Fig. 5). The linear relationship between the cell current and ethamsylate concentration in the range of 1.010 6–1.010 4 mol l 1 was obtained. The linear regression equation was i (nA)=147.8+4.53107 C (mol l 1) with a regression coefficient r=0.9995 (n=11). The detection limit estimated as the blank signal plus three times was 4.010 7 mol l 1. The precision of the proposed method shown by RSD of 0.56 % for 11 replicate determinations of 2.010 5 mol l 1 ethamsylate was good. 3.9. Sample analysis The proposed method was applied to the analysis of clinical ethamsylate tablet and injection (Yancheng Pharmaceutical Factory, Jiangsu, China), respectively. The results were compared with the spectrophotometric method recommended by China Pharmacopoeia [19]. As seen in Table 1, there were no significant differences between the proposed method and the spectrophotometric method. To study the accuracy of the proposed method and to check the interference from excipients used in the dosage forms, recovery experiments were carried out by the standard addition method. Each recovery was calculated by comparing the results obtained before and after the

Table 1 Determination and recovery results of ethamsylate in pharmaceutical formulations (n=5) Sample

Labeled (mg)

Mean value (t 0.05, 5) (mg) Proposed method

Spectrophotometry

Tablet

250

246.3F0.2

248.1F0.4

Injection

500

503.1F0.3

505.0F0.3

Added (10 5 mol l 1)

Found (10 5 mol l 1)

Recovery (%)

RSD (%)

1.00 2.00 1.00 2.00

1.03 2.10 1.01 2.08

102.5 105.0 100.8 104.0

1.9 1.6 1.7 2.1

70

J.-Q. Chen et al. / Microchemical Journal 80 (2005) 65–70

addition of ethamsylate standard solution. The results showed that the recovery was good (Table 1). Therefore, the proposed method is available for the quantitation of ethamsylate in pharmaceutical preparations.

Acknowledgements Thanks for the financial support of the Nature Science Foundation of P.R. China (Grant No. 20475043) for present work.

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