ISSN 10619348, Journal of Analytical Chemistry, 2014, Vol. 69, No. 8, pp. 769–776. © Pleiades Publishing, Ltd., 2014.

ARTICLES

Simple, Rapid and Sensitive Method for the Determination of Mefenamic Acid in Pharmaceutical Preparations1 Mouayed Q. Al Abachi and Hind Hadi* Department of Chemistry, College of Science, University of Baghdad Jadriyah, Baghdad, Iraq *email: [email protected] Received April 12, 2012; in final form, September 07, 2012

Abstract—Two simple and fast automated methods for the direct determination of mefenamic acid (MEF) in pharmaceutical samples are described. Continuous flow and stoppedflow systems with spectrophotomet ric detection of mefenamic acid with sodium nitroprusside and hydroxylamine hydrochloride were devel oped. Both methods show a good reproducibility (RSD < 1.5 and 1.8%, respectively) and a wide range of lin earity (between 10–500 and 3–300 µg/mL). The stoppedflow protocol has a lower detection limit (1.2 µg/mL) with a sensitivity of about two times greater than the continuous flow technique. The proposed procedures are rapid, reliable and can be applied successfully to the estimation of mefenamic acid in different commercial forms. Keywords: stoppedflow injection, mefenamic acid, spectrophotometry DOI: 10.1134/S106193481408005X 1

Mefenamic acid, 2[(2,3dimethylphenyl)ami no]benzoic acid is a kind of nonsteroidal antiin flammatory drug. It has antiinflammatory, analgesic and antipyretic actions and is used mainly in the treatment of rheumatoid arthritis and osteoarthritis as well as other muscularskeletal diseases [1]. Litera ture has reported a variety of methods for the determi nation of MEF as pure compound or in dosage forms. These methods include chromatographic [2, 3], titri metric [4], flow injection [5–7], spectroflourometric [8] and spectrophotometric [9–15]. However, some of these procedures suffer from one or another inconve nience such as requiring extraction [9–11], using non aqueous medium [12], low sensitivity [13, 14] as well as the need for pH control or heating [15]. Until now, very little studies have discussed continuous flow in jection analysis methods (cFIA), and there is a lack of studies on stoppedflow injection analysis methods (sFIA) for the determination of MEF in the pure form or in pharmaceutical preparations. The flow injection analysis (FIA) technique involves the injection of a sample solution into a carrier stream of a suitable re agent which flows through a narrow tube into a detec tor, whereby the resultant derivative can be measured [16]. Several detection systems were used in FIA, but UV–VIS spectrophotometry detector is most wide spread for the detection of pharmaceuticals, because of its adaptability and low cost [17, 18]. Several kinds of FIA were appeared and developed like continuous and stoppedflow injection manifolds. The cFIA re fers to measuring the concentration of the analyte un 1 The article is published in the original.

interruptedly in a stream of a flowing liquid [19]. On the other hand, sFIA involves stopping off the sample zone either in reaction coil or in flow cell. This results in improved sensitivity, increased reaction time, de creased detection limit and higher sample throughput [20]. Recently, many reports have been published about the applications of stoppedflow technique for quantitative analysis [21–23]. The aim of different kinds of flow injection methods is to analyze a maxi mum number of samples with a minimum amount of reagents, sample solution and analysis time [24]. In the current study, simple, fast and sensitive con tinuous and stoppedflow injection spectrophotomet ric methods have been described for the determination of MEF. MEF reacts immediately with sodium nitro prusside and hydroxylamine hydrochloride to form a colored product which can be detected spectrophoto metrically. Both of the flow injection methods have been utilized for the determination of MEF in some selected local pharmaceutical preparations. EXPERIMENTAL Reagents. Standard mefenamic acid stock solution (1000 µg/mL) was prepared by dissolving 0.1 g of the pure compound (provided from state company for Drug Industries and Medical Appliance, SDI, Sama ra, Iraq) in a sufficient amount of 0.1 M NaOH and di luted to 100 mL in a volumetric flask with the same solvent. Working solution (200 µg/mL) was prepared by simple dilution using the same solvent. Sodium ni troprusside solution (0.2%, w/v) was prepared by dis solving 0.2 g of sodium nitroprusside (BDH, UK) in

769

770

MOUAYED Q. AL ABACHI, HIND HADI (a) Sample A

HAH

RC W

B

SNP

IV FC

P

D

(b) T

F S Sample

A

HAH

RC W

B

SNP

IV FC

P

D

Fig. 1. Schematic diagram of flow injectionspectrophotometric analysis: a—cFIA system, b—sFIA system. Notations: IV— injection valve, RC—reaction coil, P—peristaltic pump, FC—flow cell, D—detector (VIS–spectrophotometer), W—waste, T—timer, HAH—hydroxylamine hydrochloride, SNP—sodium nitroprusside.

100 mL of distilled water. Hydroxylamine hydrochlo ride solution (20 mM) was prepared by dissolving 0.1389 g of hydroxylamine hydrochloride (Merk, Darmstadt, Germany) in 100 mL of distilled water. Sodium hydroxide (BDH, UK), 100 mM was pre pared by dissolving 0.4 g of the base in 100 mL of dis tilled water. Apparatus. The spectral and absorbance measure ments were carried out using a digital double beam re cording spectrophotometer (Shimadzu, UV–VIS 260). A flow cell with 50 µL internal volume and 1 cm path length was used for the absorbance measurements. Furthermore, twochannel manifolds were employed for the normal flow and stoppedflow injection meth ods. The system also involved the use of a peristaltic pump (Ismatec, Labortechnik Analytik, Glatbrugg, Zurich, Switzerland) in order to transport the solu tions. In addition, an injection valve (Rheodyne, Altex 210, Supelco, USA) was used to deliver appro priate injection volumes of the standard solution and samples. In addition, flexible vinyl tubing of 0.5 mm internal diameter was used for the peristaltic pump. The reaction coil of Teflon material had an internal di ameter of 0.5 mm. Flow manifolds. Flowinjection system. A twochan nel manifold was employed for continuous and stoppedflow injection spectrophotometric determi nation of MEF (Fig. 1a). Channel A was used to trans port hydroxylamine hydrochloride, while sodium ni troprusside was transported via channel B. The sample

was injected into the stream of both reagents solutions through the injection valve. Finally, the solutions were propelled by the peristaltic pump which had individual flow rate of 0.85 mL/min and the absorbance was measured at 647 nm. Stoppedflow system. A sFIA manifold for direct de termination of MEF has been developed (Fig. 1b), us ing the same twochannel manifold used in cFIA. The difference between the two systems is that the peristal tic pump interconnects with a programmed timer which allows the flow to be stopped at 18 s after each injection when the reacting mixture is in the detector flow cell. Similarly, the absorbance was measured at 647 nm. Procedure. General batch procedure. An aliquot of sample containing 20–400 µg of MEF was transferred into a series of 10 mL standard flasks. A volume of 1 mL of 20 mM of hydroxylamine hydrochloride solution or 0.7 mL of 0.2% (w/v) of sodium nitroprusside solution was added. The contents of the flasks were diluted to the mark with distilled water, mixed well and left for 30 min. The absorbance was measured at 647 nm at room temperature (25°C) against reagent blank con taining all materials except MEF. A calibration graph was drawn and the regression equation was calculated. For the optimization of conditions and in all subse quent experiments, a solution of 200 µg was used and the final volume was 10 mL. General cFIA procedure. Working solutions of MEF in the range 10–500 µg/mL were prepared from stock

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RESULTS AND DISCUSION This study examined the parameters affecting mainly the sensitivity and stability of the colored prod uct resulting from the reaction of MEF with sodium nitroprusside (SNP) in the presence of hydroxylamine hydrochloride (HAH). The effects of these parameters were established by changing one parameter at a time and keeping the others fixed and observing the effect produced on the absorbance of the colored species. These parameters were time, temperature, volume and strength of SNP, NH2OH reagents and the order of addition of reagents. The absorption spectra of the colored product are given in Fig. 2. In the current study, MEF functioned as a donor because of the pres ence of a secondary amine. In alkali medium and in the presence of hydroxylamine, sodium nitroprusside exists as aquoferrocyanide [Fe(CN)5H2O]3–. Based on the fact that electron transfer relies upon the degree of delocalization of both donor and acceptor metal orbit JOURNAL OF ANALYTICAL CHEMISTRY

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0.7 0.6 Absorbance

solutions. A 150 µL portion of MEF was injected into the stream of 20 mM sodium nitroprusside and 50 mM of hydroxylamine hydrochloride with a flow rate of 0.83 mL/min in each channel (Fig. 1a). The resulting absorbance of the green dye was measured at 647 nm. Optimization of conditions was carried out on 200 µg/mL of MEF. General sFIA procedure. The flow system had an in dividual flow rate of 1.63 mL/min (Fig. 1b) and con sisted from two stream lines. The first one was to deliv er a 30 mM of sodium nitroprusside solution which then merged with 50 mM of hydroxylamine hydro chloride stream. A 200 µL portion of MEF (working solutions in the range 3–300 µg/mL) was injected into these combined lines to form a green complex product which then directed towards the flow cell in the detec tor. The pump was stopped for 120 sec via the control unit at 18 s after each injection when the sample zone was in the detector flow cell. Then the flow was restart ed to push the zone out of the detector, and this step needed 50 s before the absorbance was monitored spec trophotometrically at 647 nm. The optimization of con ditions was carried out on 200 µg/mL of MEF. Procedure for tablets and capsules. Ten tablets were weighed and powdered by triturating, and the contents of 10 vials were mixed together. The weighed powdered material equivalent to 50 mg of the pure drug was dis solved in 50 mL of 0.1 M NaOH and the volume of the mixture was made up to the mark with the same sol vent. The flask with its contents was shacken well and filtered using filter paper. Then a sample of 100, 200 and 300 µg of MEF in a final volume of 10 mL was tak en and the measurements were carried out as de scribed earlier under general batch procedure. The other samples of 50, 100 and 200 µg/mL of MEF which were used in the FIA method were prepared by simple dilution from stock solution using the same solvent. Similarly, the measurements were carried out as described earlier under general procedures.

771

1

0.5 0.4 0.3 0.2 2 0.1 0 420

470

620 520 570 Wavelength, nm

670

720

Fig. 2. Absorption spectra of 40 µg/mL MEF treated as described under procedure and measured against reagent blank (1) and the reagent blank measured against distilled water (2).

als of the overlapping ligands. Therefore, the ligands which contain a single bond like ammonia and water are probably much less efficient in conducting elec trons between metal ions than strong and unsaturated ligands such as CN ⎯ which forms complexes of high degree of covalency and electron delocalization. Based on the analogy [25, 26], the sequence of reac tions is presented in Scheme: [Fe(CN)5NO]2– + NH2OH

Alkali

COOH H N [Fe(CN)5H2O]3– +

[Fe(CN)5H2O]3–

CH3 CH3

MEF 3–

COOH Fe(CN)5:N H

CH3 CH3

Proposed mechanism of the reaction between MEF and SNP.

The obtained results from Job’s method in Fig. 3 showed that a 1 : 1 (MEF : SNP) product was formed be tween drug and sodium nitroprusside at 647 nm. The sta bility constant of the dye product was 8.8 × 103 L/mol. Batch spectrophotometric determination. Prelimi nary experiments explained that the green dye product which was formed between MEF and SNP was more ef ficiently produced in a slightly basic medium. Moreover, it was found that the alkaline solution (0.1 M NaOH) No. 8

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MOUAYED Q. AL ABACHI, HIND HADI

0.5

Absorbance

0.4 0.3 0.2 0.1

0

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 mole MEF/[mole MEF + mole SNP]

Fig. 3. The molar ratio of the reaction between MEF and SNP.

which is used for dissolving MEF is sufficient for de veloping the colored reaction. Therefore, the effects of different kinds of basic solutions used for dissolving MEF such as sodium carbonate, sodium acetate, sodi um hydroxide and ammonia were studied. Maximum sensitivity and stability were obtained only when the MEF was dissolved in 0.1 M NaOH solution. The best experimental conditions for the determination of MEF were established in order to obtain high sensitiv ity and stability using different concentrations of HAH (from 0.6 to 5 mM) and SNP (from 0.2 to 1.7 mM) by varying one of the variables and fixing the others. The obtained results showed that 2 mM of HAH and 0.5 mM of SNP can give the highest absorption intensity as well as stability of the dye product for 200 µg of MEF in a final volume of 10 mL (20 µg/mL). The obtained green colored product is formed immediately becomes stable after 20 min and remains stable for more than 90 min. This study also showed that room temperature (25°C) is better for obtaining high absorbance of the colored compound than when the color was developed in an icebath (0°C) or in a water bath (60°C). The ef fect of some common excipients such as sucrose, glu cose, fructose, lactose, starch, talc and magnesium

stearate was studied by analyzing synthetic sample so lutions containing 20 µg/mL of MEF and excess amounts (12.5fold) of each excipient. It was shown that none of these substances interfered seriously with the reaction. The regression equation obtained from a series of MEF standards and the analytical figures of merit of this procedure are summarized in Table 1 which also summarizes the main performance of the flow pro cedures for the determination of MEF in order to make an effective comparison between the three ap proaches. FIAspectrophotometric determination (cFIA and sFIA). In order to develop the cFIA and sFIA proce dures, the batch method for the determination of MEF was adopted as a base procedure. Both mani folds used for the determination of MEF were de signed to provide different reaction conditions for magnifying the absorbance signal generated by the re action of MEF with SNP. For cFIA, maximum absor bance intensity was obtained when the sample was in jected into a stream of mixed SNP and HAH (Fig. 1a), whereas the manifold used for sFIA also involved the injection of MEF into the combined flow of SNP and HAH (Fig. 1b). This resulted in the formation of a green complex product which then was directed into the flow cell of the detector at which the pump was stopped (for 120 s at 18 s after each injection). The stoppedflow modification has increased absorbance of the reacting mixture and sensitivity. Therefore, the optimization of the time point for stopping is a very important step in sFIA method. The colored product and the absorbance were monitored spectrophotomet rically at 647 nm, and the absorbance of blank was subtracted. In the current study, all chemical, physical and hy drodynamic variables having an effect on the color re action were examined and optimized as explained be low. In all cases, samples were injected in triplicate and the results were corresponding to the average values. Effect of chemical parameters. The green colored complex formed between MEF and SNP in the pres ence of HAH is relatively stable and its intensity is un usually high. Furthermore, the blank for the reagents

Table 1. Analytical features of the procedures developed for the determination of MEF Parameter Regression equation Molar absorption coefficient, L/mol cm Linear range, µg/mL Correlation coefficient Limit of detection (S/N = 3), µg/mL Limit of quantification, µg/mL Reproducibility, % Recovery, % Throughput, h–1

Batch procedure

cFIA

sFIA

y = 0.0116x – 0.0191 2.799 × 103 2–40 0.9989 0.6 1.8 <2.5 100.1 2

y = 0.0020x – 0.0290 0.483 × 103 10–500 0.9991 3.1 10.2 <1.5 100.4 72

y = 0.0035x – 0.0419

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773

Table 2. Optimization of experimental variables Optimum value Variable

Studied range cFIA

sFIA

Sodium nitroprusside, mM

3–100

20

30

Hydroxylamine hydrochloride, mM

3–100

5

5

Reaction coil, cm

25–250

100

100

Total flow rate, µL/min

0.75–8.00

1.65

Sample volume, mL

50–250

Stopped time, s

10–300

120

Flow time after stopping injection has stopped, s

10–30

18

(i.e., in the absence of MEF) had very low absorbance at the selected wavelength. The effect of SNP concen tration on the color reaction was studied in the range of 3–100 mM for both flow injection methods. Con centrations of 20 and 30 mM gave the highest absor bance for cFIA and sFIA methods, respectively, and were chosen for further use. The effects of various con centrations of HAH in the range of 3 to 100 mM were also investigated. A concentration of 5 mM gave the highest absorbance for both methods of FIA and was chosen for further use (Fig. 4). Effect of manifold parameters. The physical param eters which were studied under the optimized reagent concentrations involved the flow rate, the injected sample volume and the reaction coil length. Coil length is an essential parameter that affects the sensi tivity of the colored reaction and was investigated in the range of 25–250 cm. The result obtained showed that a coil length of 100 cm was adequate to create an efficient mixing of both streams and gave the highest absorbance for both normal and reverse flow injection procedures (Fig. 4). It was used in all subsequent ex periments. The effect of flow rate on the sensitivity of the col ored reaction product was investigated in the range of 0.75–8 mL/min. The absorbance decreased with the increase of flow rate in cFIA because the high speed did not give the reactant a sufficient time for mixing, i.e. the sample zone would form and leave the flow cell quickly. On the other hand, in sFIA the absorbance slightly changed with increasing flow rate, this may re sult from that the stopping time in sFIA that would permit the reaction to be completed and sensitivity to increase. The results obtained showed that a total flow rate of 1.65 and 3.25 mL/min (0.8 and 1.63 mL/min in each line) for cFIA and sFIA, respectively, gave the highest absorbance (Fig. 4), which was used in all sub sequent experiments. The volume of the injected sam ple was varied between 50 and 250 µL using different lengths of sample loop. The results obtained showed that injected sample of 150 and 200 µL for both cFIA JOURNAL OF ANALYTICAL CHEMISTRY

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150

3.25 200

and sFIA, respectively, gave the highest absorbance (Fig. 4). These volumes were used in all subsequent ex periments. Optimization of the flow and stopping time period. Stoppedflow injection technique was first used to get better sensitivity and decrease the detection limit to achieve a higher sample throughput. Depending on the optimum manifolds, the flow time after each in jection (10–30 s) was studied. It was found that the sample zone reached the flow cell in 18 s after each in jection. In addition, the time intermission between stopping and restarting of the pump flow in the range of 10–300 s were also studied. The results showed clearly that the increase of stopping time would in crease the absorbance (Fig. 4) because this increment in the stopping time gives the reactants an adequate time for reaction and enhances the sensitivity of the determination. After 120 s, the increments in stopping time have little effect, which makes this time sufficient for subsequent work and selected as an optimum inter val time. The stoppedFI signal profiles obtained from applied method are shown in Fig. 5. The unvaried optimization method which was used above to study the effect of variables on the absorbance intensity that gave the optimum conditions in both continuous and stopped FIA methods are shown in Table 2. Analytical application. The proposed batch and FIAspectrophotometric methods have proved to be very simple and costeffective for determination of MEF. The developed methods are adequate for analy sis of aqueous solutions and pharmaceutical prepara tions and have the advantage of using a small amount of sample. In addition, these methods neither require previous separation step nor a temperature or a pH controller. The proposed procedures are economical, especially when compared to other methods, with a good linearity and sensitivity when compared with the batch or other FIA methods. For example, in compar ison between the batch and FIA procedures, the later method is faster (sample throughput of 72 and 24 in No. 8

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MOUAYED Q. AL ABACHI, HIND HADI 0.7

0.7 1 0.6

0.6

0.5

0.5 Absorbance

Absorbance

1 0.4 2 0.3

0.3

0.2

0.2

0.1

0.1

0

15

45 30 cSNP, mM

60

2

0

75

0.7

0.9

0.6

0.8

20

40

80 60 cNAN, mM

Absorbance

0.4

1

0.3 2

0.2

120

1

0.6 0.5 0.4 0.3 0.2

0.1

2

0.1

0

50

150 200 100 Reaction coil, cm

75250

300

1.5

0

3.0 4.5 6.0 Total flow rate, mL/min

7.5

9.0

0.50

0.75 1

0.45 Absorbance

0.65 Absorbance

100

0.7

0.5 Absorbance

0.4

0.55 0.45

2

0.40 0.35 0.30 0.25

0.35

0.20 0.15

0.25 0

50

100 150 200 Sample volume, µL

250

300

0

40

80

120 160 200 Stopped time, s

240

280

320

Fig. 4. Effect of chemical and physical parameters on the reaction between SNP and HAH; 1—sFIA, 2—cFIA.

jection/h for cFIA and sFIA, respectively), and with a wider linear range of the calibration graph (Table 1). In addition, sFIA has many advantages since it is twice more sensitive than cFIA and with a lower detection limit. The precision of the methods was evaluated by

analyzing pure sample of MEF, and a good recovery was obtained. The proposed methods were applied suc cessfully to the analysis of some pharmaceutical prepara tions containing MEF. The results in Table 3 are in accor dance with those obtained by the official method using

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775

0.9 F

0.8

S

W

0.7 1

Absorbance

0.6 0.5 0.4 2 0.3 3 0.2 4 0.1 A 0

B

C

20

40

60

80

100

120

D

140

160

180

200

Time, s Fig. 5. Stopped–FI signal profiles obtained for MEF concentrations 200 (1), 150 (2), 100 (3), 0 (4) ppm. Notations: F—flow time, the consumed time from the injection to the stopping point; S—stopping time, a period of time during which the flow is stopped for a short time; W—washing time, the consumed time for pushing the zone out of the detecting flow cell; A—injection sample point; B—point of stopping the pump; C—point of restarting the flow; D—end point of analysis.

Таблe 3. Application of proposed and official methods to the determination of MEF drug in chosen dosage forms (n = 5) Batch

cFIA

sFIA

Drug form recovery, c, µg/mL % Ponstidin® tablet (500 mg) SDI, Iraq

Ponstidin® capsule (250 mg) NDI, Iraq

Mepstan® tablet (500 mg) Unifarma, U.A.E.

RSD, %

recovery, c, µg/mL %

RSD, %

recovery, c, µg/mL %

RSD, %

10

98.29

1.2

50

100.29

0.9

50

99.19

2.2

20

99.78

0.7

100

100.34

0.9

100

99.98

2.3

30

99.58

0.8

200

99.34

0.7

200

98.38

1.1

10

100.78

1.7

50

99.25

1.2

50

101.88

2.7

20

100.28

1.4

100

98.72

0.9

100

101.58

0.9

30

101.39

1.3

200

98.10

1.4

200

99.46

1.0

10

99.17

2.0

50

100.15

1.4

50

98.83

1.1

20

100.45

1.1

100

99.74

1.0

100

100.25

1.1

30

99.16

0.7

200

101.23

1.6

200

100.69

0.9

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Official method recovery

99.88

100.50

100.20

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MOUAYED Q. AL ABACHI, HIND HADI

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