Multicommutated generation of concentration gradients in a flow-batch system for metronidazole spectrophotometric determination in drugs

Analytica Chimica Acta 511 (2004) 113–118

Multicommutated generation of concentration gradients in a flow-batch system for metronidazole spectrophotometric determination in drugs Everaldo Paulo Medeiros, Elaine Cristina Lima Nascimento, Ana Cláudia Dantas Medeiros, Jose Germano Veras Neto, Edvan Cirino da Silva, Mario Cesar Ugulino Araujo∗ Departamento de Quimica, Universidade Federal da Paraiba, Caixa Postal 5093, CEP 58051-970, Joao Pessoa PB, Brazil Received 22 August 2003; received in revised form 16 January 2004; accepted 20 January 2004

Abstract The titration by using the perchloric acid as titrant and malachite green as indicator is the classical method for the determination of metronidazole, 2-(2-methyl-nitroimidazol-1-yl) ethanol, in drugs. However, this procedure is slow, laborious and consumes great amount of reagents and samples. Moreover, it requires careful manipulation because the reagents cause irritation in the skin and in the eyes. To overcome these drawbacks an automatic system is proposed. It uses three-way solenoids valves, an open chamber and it exploits concentration gradients generated into the chamber in a multicommutated way. As the proposed system embodies the characteristics of flow-batch systems and of multicommutation techniques in flow, it is here named flow-batch-multicommutated titrator. The system is fully controlled by microcomputer running software written in LabView 5.1 graphic language. It processes 60 samples per hour with relative standard deviation smaller than 2.3% (n = 6) and in each titration it consumes 2.5 and 1.5 ml of sample and titrant, respectively. By applying the paired t-test, no statistic differences at the 95% probability level between the results of the proposed system and classical titration were observed. © 2004 Elsevier B.V. All rights reserved. Keywords: Concentration gradient; Multicommutation; Flow-batch system; Metronidazole; Drugs; Spectrophotometric titration

1. Introduction Metronidazole, 2-(2-methyl-nitroimidazol-1-yl) ethanol, is a substance that has a wide range of uses due to its activity against protozoa and anaerobic bacteria [1]. The titration by using the perchloric acid as titrant and malachite green as indicator is the classical procedure for the determination of metronidazole in drugs [2]. However, this classic titration when accomplished by manual procedure is slow, laborious and it consumes great amount of reagents which require careful manipulation because they can cause irritation in the skin and in the eyes. These drawbacks can be overcome if metronidazole titration is performed by a automatic procedure in flow but up to now no system was described in literature. To implement automatic titration in flow, several strategies were described [3–24] and those requiring calibration ∗ Corresponding author. Tel.: +55-83-216-7438; fax: +55-83-216-7437. E-mail address: [email protected] (M.C.U. Araujo).

0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.01.031

based on the titration of synthetic samples [3–13] present the following drawbacks: (a) difficulty in preparing the synthetic samples, e.g., in the determination of hardness, oxidability or acidity/alkalinity in natural waters, might limit the applicability of these systems; (b) results may be dependent of more than one analyte concentration, and different analytes may present different reaction stoichiometries towards the titrant, so that preparation of the standard solutions may become cumbersome. Among the proposed strategies that do not request calibration based on the titration of synthetic samples [14–24], there are two where the automatic titration was implemented of a fast way when concentration gradients generated in flow injection systems were exploited [17,20]. However, these systems present a considerable limitation to analyze samples with high variability in the analyte concentration due to necessity of modification in the flow manifold to adjust the adequate relation of instantaneous concentration between sample and titrant at the detection point. In this work, a novel strategy is proposed to automate in a fast, flexible and versatile manner the classical method

114

E.P. Medeiros et al. / Analytica Chimica Acta 511 (2004) 113–118

for the determination of metronidazole in drugs. For that, the titration approach is implemented by using a system here named of flow-batch-multicommutated titrator because it combines the inherent favorable characteristics of the flow-batch system [18] and multicommutation technique [25]. The flow-batch systems are characterized by the use of three-way solenoids valves [18] or multi-port selecting valve [26–28] and an open chamber. In these systems, the sampling, additions of reagents and signal monitoring are done in the same way as in flow analyzer, whereas the mixing, gradient generation and reaction are performed inside an open chamber as in batch systems. The concentration gradients are generated when the sample and the indicator are added in a multicommutated way into the chamber previously filled with titrant while the product of the reaction is aspirated towards the detector.

2. Experimental 2.1. Reagents, samples, and solutions The indicator solution (1.0 g l−1 malachite green in acetic acid medium) and the titrant (perchloric acid, 9.12 mmol l−1 in acetic acid/acetic anhydride medium) were prepared as described in the literature (Section 2.4). The dye solution (0.6 g l−1 malachite green) and a pH = 4.0 buffer (acetic acid/sodium acetate) were used only gradient calibration step. Samples were prepared triturating 10 metronidazole tablets in mortar and pestle. About 300 mg of sample was weighted, dissolved in acetic anhydride and then filtered and transferred into a 100 ml volumetric flask. The volume of flask was completed with acetic anhydride. All chemicals were of analytical grade quality. The buffer and the dye solution were always prepared with freshly distilled-deionised water. The flow-batch-multicommutated titration was always carried out in acetic acid medium.

2.2. Flow-batch-multicommutated titrator A schematic diagram of the developed titrator is depicted in Fig. 1a. A model B342II Micronal spectrophotometer furnished with a tubular (inner volume 100 ␮l and optical path 2 mm) flow cell [29] and operated in 623 nm wavelength was employed as detector. A model MCP Ismatec peristaltic pump equipped with five pumping channels and employing a 2.0 mm i.d. silicone pumping tubes was used. The transmission lines linking the valves VS , VT , VI , and VD to GC and VD to detector were implemented as short as possible using 0.8 mm i.d. Teflon® tubing. The gradient chamber (GC) was made from Teflon® in a cylindrical shape with an inner diameter of 0.9 cm and an inner volume of about 2.2 ml (Fig. 1b). A model 190 M Hanna Instruments magnetic stirrer (MS) driven a 0.7 cm stirring bar (SB) inside the gradient chamber. Four Cole–Parmer three-way solenoid valves were used: VS , VT , and VI to direct the sample, titrant, and indicator to the GC, respectively, and VD to select the stream flowing (air or GC mixture or AA) through the detector. An MMX-Pentium 233 MHz microcomputer equipped with a laboratory-made interface card and running a program in LabView 5.1 graphic language was used to control the flow-batch-multicommutated system, analytical signal acquisition and data treatment. The solenoids valves and magnetic stirrer were controlled through an electronic actuator (EA), which provided the required increase in power (potential and/or current) for signal sent by the microcomputer. 2.3. Flow-batch-multicommutated analysis To implement the flow-batch-multicommutated analysis two steps are inherent: gradient calibration and titration. The gradient calibration step is needed to find the instantaneous concentrations at the detection point, in the absence of any reaction, of the sample whose concentration gradients are

Fig. 1. (a) Diagram of the flow-batch-multicommutated system: GC = gradient chamber; MS = magnetic stirrer; VT , VS , VI , and VD = solenoid valves; T = titrant; S = sample; I = indicator; BS = buffer solution; D = detector; EA = electronic actuator; PC = microcomputer. The arrows indicate the direction of pumping and aspiration of the fluids. (b) Dimensions of GC (values in cm). SB = stirring bar.

E.P. Medeiros et al. / Analytica Chimica Acta 511 (2004) 113–118

yield and exploited during the titration procedure. As shown in Section 2.5, the instantaneous concentrations are obtained associating the time in gradient concentration profile to a dilution-like factor that defines a relationship between the original concentration of the sample in the flask and the instantaneous concentrations at the detection point. The gradient calibration procedure is made, in the absence of any reaction, by using only one solution containing a specimen capable to be sensed by the detector and producing a response linearly related with its concentration. In this work, a dye solution (0.6 g l−1 malachite green) was utilised for this purpose. Before starting gradient calibration or titration procedure, the working solutions of each channel is pumped and recycled towards its flasks. After, the valves (VS , VT , and VI ) are simultaneously switched ON during a time interval of 2 s and the working solutions are pumped towards GC to fill the channels between the valves and GC. Right after, VD is switched ON and the excess of the solutions into GC are aspirated to waste during 3 s. This process is named here ‘channels filling’ and it should always be accomplished when the solution of each channel is changed. After finishing the steps of gradient calibration, titration or channels filling, the procedure of GC cleaning is always performed. VT is switched ON and the cleaning solution (buffer or titrant) is pumped into GC during 5 s. After, VD is switched ON and the GC content is discarded. This procedure is always performed two times in order to guarantee a more efficient cleaning. 2.3.1. Gradient calibration procedure Initially, the baseline signal (BS, Fig. 2) is obtained aspirating buffer solution via VD towards detector. After, the steady state signal (A0dye , Fig. 2a) is recorded when dye solution is also aspirated via VD during 5 s. Before gradient calibration, the procedure of channels filling is accomplished by pumping dye solution in the channels of sample and indicator, and buffer solution in the channel of titrant. VT valve is then switched ON during the time interval of 20 s to fill GC with buffer solution which is aspirated during 5 s. Filled GC, the VD valve is switched ON

115

and GC content is aspirated towards the detector while the dye solution is pumped in multicommutated manner into GC during 35 s by alternately switching VS and VI valves ON/OFF at short time intervals of 300 ms. Thus, a concentration gradient of dye solution is generated in flow-batch system and a linear curve (absorbance × time) is recorded (AG dye (t), Fig. 2b). 2.3.2. Titration procedure Initially, the sample, the indicator (1.0 g l−1 malachite green) and the titrant (9.12 mmol l−1 HClO4 ) are pumped in its channels and recycled into its flasks (Fig. 1a). After the channels filling process, VT valve is switched ON during 20 s to fill GC with titrant. Right after, the sample and indicator are pumped in multicommutated way into GC as in the gradient calibration procedure, whereas the product of the titration reaction is aspirated towards the detector. As a result, the signal AG S1 (t) illustrated in Fig. 2c is obtained. Fig. 2d and e illustrates the recorded signals in the titration of two other samples with different concentrations. However, before accomplishing the titration of a new sample, the cleaning of GC should be performed. For that, the same procedure described in the calibration step is repeated, but the titrant should be used instead of the buffer solution. The VD valve was always maintained switched ON during the measurements of analytical signals. 2.4. Reference procedure The metronidazole determinations were also performed using the reference procedure recommended by the United States Pharmacopoeia [2] in order to assess the efficiency of the proposed flow-batch-multicommutated methodology. 2.5. Theoretical If Beer’s lay is obeyed, the steady state signal (Fig. 2a) of dye solution can be calculated as: A0dye = kC0dye

(1)

Fig. 2. Illustration of the obtained signals in flow-batch-multicommutated analysis: (a) and (b) signals of the gradient calibration step; (c), (d), and (e) signals for consecutive determination of three samples with different concentrations. For details and symbol description, see text.

116

E.P. Medeiros et al. / Analytica Chimica Acta 511 (2004) 113–118

0 is the original known concentration of the dye where Cdye solution in its flask and k is the response linear constant. The flow-batch-multicommutated system is designed to generate a concentration gradient profile of dye solution. Thus, if Beer’s lay is obeyed, a linear curve between the instantaneous absorbance of the dye solution,AG dye (t), and time t, is recorded in microcomputer (Fig. 2b) and the following equations can be written: G 0 Cdye (t) = gdye (t)Cdye

(2)

and G AG dye (t) = kCdye (t)

(3)

where, g(t) is a dilution-like factor that defines a relationship between the original concentration of the dye solution in the 0 , and the instantaneous concentrations, C G (t) its flask, Cdye dye of this same solution at the detection point. By combining the Eqs. (1)–(3), the following expression is obtained: gdye (t) =

AG dye (t) A0dye

(4)

By assuming that the concentration gradient profile of dye solution (Fig. 2a) is identical [17,20] to the concentration gradient profile of the analyte during flow-batchmulticommutated titration (Fig. 2c–e), the Eqs. (2) and (4) can be used to deduce the following expression: G CA (t) =

AG dye (t) A0dye

0 CA

(5)

0 is the unknown analyte concentration in the samwhere, CA G (t) is the instantaneous concentrations of this ple and CA same solution at the detection point. The method here described makes use of the stoichiometric instantaneous point (SIP) [17,20] and this point is located between regions where excess of titrant and analyte always occurs. At this point, the instantaneous concentration of the G (t analyte, CA SIP ), is at stoichiometric ratio in relationship to the titrant concentration, CT0 , added into GC. Thus, at the SIP, the following relationship is valid: 0 yCG A (tSIP ) = xCT

(6)

where x and y are the stoichiometric coefficients of the titration reaction (xA + yT → Ax Ty ) between the analyte and the titrant. By substituting Eq. (6) in Eq. (5), the unknown analyte 0 , can be calculated by: concentration in the sample, CA 0 CA

0 x Adye CT0 = y AG dye (tSIP )

(7)

2.5.1. SIPs obtainment SIPs should be obtained in curves where abrupt changes in the propriety being observed (absorbance, for example)

G G Fig. 3. AG S (t) × Adye (t) curve (in arbitrary units of absorbance). Adye (tSIP )

is the AG dye value at the stoichiometric instantaneous points (SIP).

are plotted against the instantaneous concentrations of the analyte. As AG dye (t) values can be related to the instantaneous concentrations of the analyte (see Section 2.5), SIPs G can be found in AG S (t) × Adye (t) curves. These curves are constructed by replacing t in AG S (t)×t curves (Fig. 2c–e) for its equivalent AG (t) values in AG dye dye (t) × t curve (Fig. 2b), at the same time. This procedure ensures that the changing propriety is being evaluated against the instantaneous concentrations of analyte in a similar way as conventional instrumental titrations are evaluated for end point determination in a curve of the propriety change versus added amount variation of analyte, keeping the titrant amount G constant. Fig. 3 illustrates as AG S (t) × Adye (t) curves are constructed and where SIPs are located (at the crossing point of the two straight lines observed after and before the G abrupt change of AG S (t) × Adye (t)). The software uses the interception of straight lines equations to obtain AG dye (tSIP ).

3. Results and discussion Since the total volume of GC is 3.5 ml, a 2.5 ml min−1 flow-rate was selected for each channel in order to achieve a low consumption of sample and reagents, as well as a good sample throughput and precision of measurements. Moreover, higher flow rates were selected for the emptying steps of GC, in order to minimize the time required for processes of the titration. The uncertainties resulting from the solutions delivery by the solenoids valves were studied employing 1.8 ml min−1 flow-rate. Water was delivered into weighing bottles at time intervals in the range of 100–1000 ms with increments of 100 ms and its mass were measurements in analytical balance. The relative standard deviations for 10 replicate measurements corresponding at each time intervals were generally smaller than 1.0%. Besides allowing a larger versatility in the implementation of titration procedure, the gradient chamber allow to guar-

E.P. Medeiros et al. / Analytica Chimica Acta 511 (2004) 113–118 Table 1 Results (in mg per tablet) of the metronidazole determinations by the proposed method and by classical titration Sample

Classical titration

Proposed methoda

1 2 3 4 5 6 7

251 393 256 259 412 256 260

257 411 252 256 416 263 257

a Relative standard deviations of results were estimated as 2.3% (n = 6).

antee the equality between the concentration gradient profile of the dye solution and the concentration gradient profile of the analyte during flow-batch-multicommutated titration. This occurs because the transport of matter is predominantly produced by convection instead of diffusion which, if present, would be dependent of diffusion coefficients of the specimens and of the overall composition of the medium. To demonstrate the usefulness of the proposed strategy, the determination of metronidazole in drugs was elected. For comparative purposes, this analyte was also determined by using the reference procedure (Section 2.4) and both the results are shown in Table 1. A good agreement between the obtained values with proposed system and manual titration may be observed. In fact, no statistic difference at the 95% probability level was verified between the results when the paired t-test was applied. An overall relative accuracy of about 1.0% and relative standard deviation smaller than 2.3% (n = 6) were obtained. The proposed flow-batch-multicommutated titrator processes 60 samples per hour and in each titration consumes about 0.7 and 1.5 ml of sample and titrant, respectively. Different experimental designs can be performed in a flexible enough manner, simply by changing time configurations (tS and tT ) in the menu of the software developed for the flow-batch-multicommutated titrator. Thus, the dilution factor of the samples can be easily altered via software, if the expected analyte concentration varies in a larger range, without the necessity of modification in the physical part of the flow manifold. Moreover, in contrast of the previous systems [17,20], the proposed titrator allows to perform the optimization of the variables rapid, flexible, versatile manners, simply by changing configurations in the menu of the software, not by impairing the course of the determinations in large scale routine analyses.

4. Conclusions The proposed flow-batch-multicommutated system was applied successfully to automatized, flexible, and versatile determination of metronidazole in drugs. The largest flexibility and versatility of the proposed system occur because it

117

combines intrinsic favorable features of the batch, flow and multicommutation techniques. Therefore, these characteristics makes the proposed system more flexible and versatile that previous systems [17,20] based on the exploitation of gradients generated in flow injection analysis. In addition, it could be applied to the determination of hardness, oxidability or acidity/alkalinity due to the non-necessity analytical curves based on the titration of standard solutions. Finally, the possibility of utilization of the proposed system for implementing of fast ‘screening’ procedures aiming at determination of concentration range of analytes (e.g., phosphate, nitrite, etc.) in samples of natural waters with high variability in the concentration is presently under investigation.

Acknowledgements E.C.L. Nascimento and M.C.U. Araujo, and E.P. Medeiros are honoured with CNPq and CAPES scholarships, respectively.

References [1] M. Palomeque, J.A. Garc´ıa Bautista, J.V. Garc´ıa Mateo, J. Martinez Calatayud, Anal. Chim. Acta 401 (1999) 229. [2] The United States Pharmacopoeia, The National Formulary, USD XXII, 1990. [3] W.J. Blaedel, R.H. Laessig, Anal. Chem. 36 (1964) 1617. [4] B. Fleet, A.Y.W. Ho, Anal. Chem. 46 (1974) 9. [5] G. Nagy, Z. Feher, K. Toth, E. Pungor, Anal. Chim. Acta 91 (1977) 87. [6] J. Ruzicka, E. Hansen, H. Mosbaek, Anal. Chim. Acta 92 (1977) 235. [7] O. Astrom, Anal. Chim. Acta 97 (1978) 259. [8] S.M. Abitch, Anal. Chim. Acta 114 (1980) 247. [9] N. Ishibashi, T. Imato, Fresenius Z. Anal. Chem. 323 (1986) 244. [10] J.M. Calatayud, P.C. Falco, R.M. Albert, Analyst 112 (1987) 1063. [11] D.P. Arnold, R.M. Peachey, J.D. Petty, D.R. Sweatman, Anal. Chem. 61 (1989) 2109. [12] T.J. Cardwell, R.W. Cattrall, G.J. Cross, G.R. O’Connell, J.D. Petty, G.R. Scollary, Analyst 116 (1991) 1051. [13] J.M.A. R´ıos, M. Valcárcel, Anal. Chim. Acta 261 (1992) 489. [14] C.M. Yarnitzky, N. Klein, O. Cohen, Talanta 40 (1993) 1937. [15] M. Korn, L.F.B.P. Gouveia, E. Oliveira, B.F. Reis, Anal. Chim. Acta 313 (1995) 177. [16] L. Alerm, J. Bartroli, Anal. Chem. 68 (1996) 1394. [17] M.C.U. Araújo, A.V. Santos, R.S. Honorato, C. Pasquini, J. Autom. Chem. 19 (1997) 157. [18] R.S. Honorato, M.C.U. Araujo, R.A.C. Lima, E.A.G. Zagatto, R.A.S. Lapa, J.L.F.C. Lima, Anal. Chim. Acta 396 (1999) 91. [19] R.S. Honorato, M.C.U. Araujo, G. Veras, E.A.G. Zagatto, R.A.S. Lapa, J.L.F.C. Lima, Anal. Sci. 15 (1999) 665. [20] E.N. Gaião, R.S. Honorato, S.R.B. Santos, M.C.U. Araujo, Analyst 124 (1999) 1727. [21] R.S. Honorato, E.A.G. Zagatto, R.A.C. Lima, M.C.U. Araujo, Anal. Chim. Acta 416 (2000) 231. [22] C. Almeida, R.A.S. Lapa, J.L.F.C. Lima, E.A.G. Zagatto, M.C.U. Araujo, Anal. Chim. Acta 407 (2000) 213. [23] B. Fuhrmann, U. Spohn, Anal. Chim. Acta 282 (1993) 397.

118

E.P. Medeiros et al. / Analytica Chimica Acta 511 (2004) 113–118

[24] I.L. Garc´ıa, P. Vinas, N. Campillo, M.H. Córdoba, Anal. Chim. Acta 308 (1995) 67. [25] F.R.P. Rocha, B.F. Reis, E.A.G. Zagatto, J.L.F.C. Lima, R.A.S. Lapa, J.L.M. Santos, Anal. Chim. Acta 468 (2002) 119. [26] J.M.T. Carneiro, R.S. Honorato, E.A.G. Zagatto, Fresenius J. Anal. Chem. 368 (2000) 496.

[27] R.S. Honorato, J.M.T. Carneiro, E.A.G. Zagatto, Anal. Chim. Acta 441 (2001) 309. [28] J.M.T. Carneiro, A.C.B. Dias, E.A.G. Zagatto, R.S. Honorato, Anal. Chim. Acta 455 (2002) 327. [29] M.C.U. Araújo, R.S. Honorato, A.V. Santos, E.C. Silva, Quim. Nova 19 (1996) 86.