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Quantitative analysis of sulfadiazine using photochemically induced fluorescence detection in bulk solution and in a flow injection system
ANALYTICA CHIMICA ACTA Analytica
Chimica
Acta 314 (1995) 45-50
Quantitative analysis of sulfadiazine using photochemically induced fluorescence detection in bulk solution and in a flow injection system * J.J. Aaron ay*, M.I. Acedo Valenzuela b, M. Sanchez Pena b, F. Salinas b, M.C. Mahedero b a Institut de Topologie et de Dynamique des Sys&mes de l’Universit6 Paris 7 - Denis Diderot, associk au CNRS URA 34, 1, rue Guy de la b Department ofAnalytical
Brosse, 75005 Paris, France Chemishy, Faculty ofSciences, University of Extramadura, 06071 Badajoz, Spain
Received 28 November
1994; revised 4 April 1995; accepted 9 May 1995
Abstract Room-temperature photochemically-induced fluorescence (RTF’F) methods, using bulk solution and flow-injection analysis (FIA) were proposed for determining sulfadiazine (SDZ). The RTPF determination was based on the irradiation of SDZ with ultraviolet light. A methanol-water (50:50, v/v) mixture was found to be the optimal solvent for the rapid, precise, and sensitive RTPF analysis of SDZ. Limits of detection were 40 ng/ml (bulk solution) and 90 ng/ml (FL4). The methods were applied for the determination of SDZ in pharmaceuticals. Satisfactory recoveries, ranging between 93 and 111.8%, were obtained. Keywords: Fluorimetry; Flow injection; Sulfadiazine
1. Introduction Due to their increasing importance in medicine and veterinary practice, sulfonamides have been the subject of a number of analytical studies. The fluo-
* Presented, in part, at the VIth International Symposium on Luminescence Spectroscopy in Biomedical Analysis - Detection Techniques and Applications in Chromatography and Capillary Electrophoresis, Bruges, Belgium, June 5- 7, 1994. The majority of papers presented at this symposium have been published in Anal. Chim. Acta, Vol. 303, No. 1. Corresponding author. l
0003-2670/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0003-2670(95)00254-S
rescence of several sulfonamides has been investigated, and some fluorimetric methods have recently been proposed as a detection means in high-performance liquid chromatography (HPLC) D-51. However, the use of these techniques has been limited in the case of nitrogen heterocyclic sulfonamides, which generally present a weak fluorescence. Studies on the photochemical decomposition of sulfonamides were reported a few years ago [6,7]. More recently, it was shown by us that room-temperature photochemically induced fluorescence (RTPF) of several heterocyclic sulfonamides could be used to determine these compounds at the ppb level in
46
J.J. Aaron et al./Analytica
aqueous solution [8]. Ultraviolet (UV) irradiation times required for obtaining maximum fluorescence signals of the photoproducts were between 10 and 30 min. These irradiation times can be significantly reduced by utilizing non-aqueous alcoholic media
[91. On the other hand, photochemical fluorescence detectors have been mainly applied to HPLC [lO,ll], but they have been used less frequently for improving the fluorescence detection of analytes in simple hydrodynamic systems such as flow-injection analysis (FIA) [12-181. For example, Mahedero and Aaron have proposed a FIA-RTPF method for determination of sulfamethazine [15]. Sulfadiazine (SDZ), is an heterocyclic sulfonamide which is currently used in the treatment of urine infections and for ophthalmology applications. Several methods such as HPLC [19], and RTPF in aqueous medium [8], have been proposed for its determination. In this paper, we investigated the effects of alcohols and alcohol-water mixtures on the photochemitally induced fluorescence intensity of sulfadiazine. The proposed RTPF methods, using bulk solution and FIA have been applied to the determination of sulfadiazine in pharmaceutical preparations, and the results have been compared with those obtained by HPLC.
2. Experimental 2.1. Apparatus A Kontron Model SFM-25 spectrofluorometer interfaced with a Geocom microcomputer was used for fluorescence measurements in both bulk solution and FIA methods. An Osram 200 W high-pressure mercury arc lamp with an Oriel Model 8500 power supply was used for photochemical studies in bulk solution. The FIA-photochemical fluorescence system has been described previously [15]. 2.2. Reagents Sulfadiazine (SDZ) was purchased from Sigma. Pharmaceutical preparations of sulfadiazine were a generous gift of Lab. Doms-Adrian, (Courbevoie,
Chimica Acta 314 (1995) 45-50
France; Adiazine, composition per tablet: 500 mg sulfadiazine, excipient, q.s.1 and of Lab. Astra-Ifesa (Barcelona, Spain; Triglobe, composition per tablet: 180 mg trimetoprim, 820 mg sulfadiazine, excipient q.s.). Standard stock solutions of SDZ (250 pg ml-‘) were prepared by dissolving the compound in methanol (Merck, spectroscopic grade). All SDZ samples were prepared taking convenient aliquots from standard solutions and diluted to the mark in a calibrated flask with water, methanol, ethanol (Merck, spectroscopic grade), 2-propanol (Merck, analytical grade) or methanol-water mixtures. The methanol-water mixtures were prepared just before use at room temperature to avoid contraction of volume and heat releasing. 2.3. Procedure For the bulk solution method, an aliquot of the SDZ sample containing between 0.1 and 4.1 pg ml-l in a methanol-water (50:50, v/v) mixture was placed in a l-cm quartz cuvette and irradiated at room temperature with the high-pressure mercury lamp for 2.5 min. Fluorescence intensity measurements were performed at the excitation and emission analytical wavelengths of 295 and 352 nm, respectively. Linear calibration graphs were established with the SDZ solutions of known concentrations treated in the same conditions as the pharmaceutical samples. Triplicate measurements were done in all cases. In the case of the FIA method [15] a water carrier, a 4.2-ml min-’ flow rate, a 300-~1 injected volume, and a 250-cm photoreactor length were used. Samples containing between 0.2 and 6.5 pg ml-’ of SDZ in methanol-water (50:50, v/v) were injected. No cleaning up procedure was used prior to FIA analysis. Fluorescence intensity measurements were obtained with excitation and emission analytical wavelengths of 295 and 352 nm, respectively. Linear calibration graphs were established with SDZ solutions of known concentrations treated in the same way as the pharmaceutical samples. All measurements were performed in triplicate. For the applications to Adiazine and Triglobe pharmaceutical preparations, three tablets of each compound were measured by weight and powdered. The solutions of these drugs were prepared by dis-
J.J. Aaron et al. /Analytica
solving 25.9 and 11.1 mg of each compound, tively, in a lOO-ml volume of methanol.
respec-
47
Chimica Acta 314 (1995) 45-50 Table 1 Effect of alcohols on the photochemically properties of sulfadiazine
induced
fluorescence
3. Results and discussion
Previously, we used an optimal UV irradiation time of 18 min for determining SDZ in aqueous solution by RTPF [8]. In order to examine if it was possible to reduce significantly this irradiation time to maximum fluorescence value - corresponding signal of the photoproducts - we investigated the effect of alcoholic solvents on the photochemically induced fluorescence properties of SDZ. The alcohols assayed were methanol, ethanol, and 2-propanol. Fig. 1 shows the effect of W irradiation time on the fluorescence intensity of SDZ at maximum emission wavelength in different alcoholic media. All curves indicate an important increase of fluorescence signal with time. In all instances, the excitation wavelength was fixed at 295 nm. The fluorescence properties of SDZ in the various solvents under study are compared in Table 1. A slight red shift of the SDZ emission wavelength was noted upon going from methanol to ethanol and 2-propanol, and from
a Optimal irradiation Fence intensity. Maximum relative concentration of SDZ where it was 1.04 pg
0
1 .o
2.0
Tirr
3.0
4.0
(min)
Fig. 1. Effect of the ultraviolet irradiation intensity of SDZ in various alcohols.
time on the fluorescence
420 108 115 -
time corresponding
to the maximum
fluores-
fluorescence intensity. In all instances, the was 2.08 pg ml-‘, except in water medium ml-‘.
the alcohols to water. Photochemically induced fluorescence intensity maxima (I,,,) were larger in aqueous medium than in alcohols. Also, it can be seen that the optimal irradiation time ranges correwere much shorter in alcoholic sponding to I,,,, solvents (0.5-4 min) than in water (8-18 min) (see Table 1 and Fig. 1). In order to improve the rapidity and sensitivity of the RTPF method, it is desirable to use shorter irradiation times and to obtain a larger fluorescence signal. Therefore, we decided to investigate the effect of varying the alcohol percentage of
01 5.0
8-18 OS-l.5 l-2 1.5-4
361 345 350 354
Water Ethanol Methanol 2-propanol
3.1. Effect of the alcohols
I
,
1
,
I
,
I
,
2
Tir’r (mi;)
’
Fig. 2. Effect of the ultraviolet time on the fluorescence intensity of SDZ in methanol:water mixtures with various methanol percentages.
48
J.J. Aaron et al. /Analytica
Table 2 Effect of the methanol fluorescence properties
Chimica Acta 314 (1995) 45-50
3.2. Determination percentage of SD2
% methanol ’
on the photochemical b ?iin,
I max c
2-3 2.2-3.5 4.5-6.5
280 530 420
;;) 347 352 354
70 SO 30
a Percentage of methanol (v/v) in the methanol-water mixture. b Optimal irradiation time range. ’ Maximum relative fluorescence intensity. In all instances, the concentration of SD2 was 1.04 J.J~ ml-‘.
alcohol-water mixtures on these fluorescence parameters. For this study, methanol was chosen since it provided an irradiation time smaller and a fluorescence signal larger than other alcohols. Fig. 2 and Table 2 show that an increase of methanol percentage from 30 to 70% produces a marked decrease of irradiation time, while the fluorescence intensity maximum is larger for a methanol-water (5050, v/v) mixture. The larger fluorescence signal found in the latter medium compared to 30% and 70% methanol media can be attributed to a specific and enhanced solvation of the SDZ photoproduct singlet excited state in this particular mixture. Therefore, the methanol-water (50:50, v/v) medium was considered as optimal for the RTPF determination of SDZ in bulk solution. Moreover, an optimum irradiation time value of 2.5 min was selected for all analytical measurements.
Table 3 Analytical
figures of merit for the RTPF determination
Method
Concentration
range
Regression
of sulfadiazine
in bulk solutions
induced
of sulfadiazine equation b
( pg/mll
A
B
Bulk solution
0.1-4.1
FIA
0.2-6.5
26.5 (4.7) = 26.8 (4.3) =
234.7 (2.2) c 84.3 (1.3) c
The analytical figures of merit for the determination of SDZ in methanol-water (50:50, v/v) are given in Table 3. A linear calibration plot was obtained between SDZ concentrations of 0.1 and 4.1 pg/ml. The correlation coefficient was close to unity. The limit of detection (LOD) value was rather low (40 ng/ml), although it was higher than that obtained previously for the determination of SDZ in water 181. The relative standard deviation (RSD) value was particularly small (1.7% for a SDZ concentration of 2.6 pg/ml), indicating an excellent reproducibility of RTPF measurements in bulk solutions. 3.3. Determination
of suljadiazine
in a FIA system
The influence of FL4 parameters on the RTPF signal of SDZ was investigated. The flow-rate, photoreactor length, and injected volume were optimized to provide maximum RTPF response and minimum band broadening, as in our previous studies [15,18]. The optimal values for these three FIA parameters were found to be 4.2 ml/min, 250 cm and 300 ~1, respectively. Table 3 summarizes the analytical figures of merit. A linear calibration graph was established over more than one order of magnitude, with a correlation coefficient of 0.999. The LOD value (90 ng/ml) is rather low, corresponding to an absolute detectable amount of 27 ng. The RSD value was rather small (2.1% for a SDZ concentration of 2.1
in a 50% (v/v> water-methanol Residual standard deviation .Sy/x
mixture a
LOD d
LOQ ’
RSD *,s
(rig/ml)
(ng/ml)
(%o)
14.3
40
150
12.7
90
310
1.7 5.2 2.1 4.5
* s * s
A, = 295 mm; A,, = 352 mm; tirr = 2.5 min. Ref. 1201 was used for the calculation of statistical data. I, = A + B . c. IF = relative fluorescence intensity; c = analyte concentration ( pg/ml). Correlation coefficient = 0.999 in both cases. Estimated standard deviation of A and B values. LOD = limit of detection, defined as the concentration of analyte giving a signal-to-noise ratio of 3. LOQ = limit of quantification, defined as the concentration of analyte giving a signal-to-noise ratio of 10. RSD = relative standard deviation for a SDZ concentration of 2.6 pg/ml (bulk solution) and 3.1 pg/ml (FL41 (n = 10). RSD for a SDZ concentration of 0.5 pg/ml (n = 10).
J.J. Aaron et al. /Analytica Table 4 Determination tion method)
of SDZ in pharmaceutical
Adiazine * added (ml) 0.3 0.3 0.3 0.3 0.3 0.2 0.2 0.2 0.2 0.2 Triglobe b added (ml) 0.4 0.4 0.4 0.4 0.4 0.6 0.6 0.6 0.6 0.6
preparations
(bulk solu-
Table 5 Determination method)
of
SDZ
49
in
pharmaceutical
preparations
SDZ added
SDZ found
Recovery
SDZ added
SDZ found
Recovery
(ppm)
(ppm)
(o/o)
(ppm)
(ppm)
(%I
-
0.64 2.48 3.03 3.54 1.76 2.81 3.24 3.63 4.68
-
2.00 2.57 3.31 3.64 4.01 1.43 1.91 2.63 3.07 3.46
0.54 1.07 1.61 2.15 0.54 1.07 1.72 2.15
-
1.63 2.21 2.87 3.37 3.76 2.42 2.71 3.02 3.40 3.59
0.52 1.03 1.55 2.06 0.31 0.69 0.93 1.24
Adiazine a added (ml) 0.1 0.1 0.1 0.1 0.2 0.2 0.2 0.2 0.2 Triglobe b added (ml) 0.4 0.4 0.4 0.4 0.6 0.6 0.6 0.6
101.2 107.8 100.8 96.6 96.9 105.2 97.4 96.6
_ 103.0 107.8 106.0 101.8 99.3 97.1 101.6 98.1
a Adiazine solution prepared dissolving 25.9 mg of pharmaceutical preparation in 100 ml of methanol. Milliliters added to a 25-ml calibrated flask. b Triglobe solution prepared dissolving 11.1 mg of pharmaceutical preparation in 100 ml of methanol. Milliliters added to a 25-ml calibrated flask.
pg/ml), showing the FIA method. 3.4. Analytical
Chimica Acta 314 (1995) 45-50
a satisfactory
reproducibility
of
applications
The proposed RTPF methods were applied for the determination of SDZ in pharmaceutical preparations. In all cases, the standard addition procedure was used. Typical results are shown in Tables 4 and 5, for bulk solution and for FIA methods, respectively. Satisfactory recovery values were obtained, ranging from 96.6 to 107.8% for bulk solution methods, and from 92.9 to 111.8% for the FIA method. The absolute weights of SDZ contained in the pharmaceutical preparations were measured directly by both methods, and validated by the HPLC method
1.58 2.11 2.63 1.05 1.58 2.14 2.63
-
111.8 110.1 108.2 _ 99.8 96.8 92.9 106.4
-
1.50 2.40 3.07 3.50 2.38 2.71 2.96 3.29
1.03 1.55 2.06 _ 0.31 0.62 0.93
(FIA
94.9 100.5 98.1 100.4 98.6 99.4
a Adiazine solution prepared dissolving 25.9 mg of pharmaceutical preparation in 100 ml of methanol. Milliliters added to a 25-ml calibrated flask. b Triglobe solution prepared dissolving 11.1 mg of pharmaceutical preparation in 100 ml of methanol. Milliliters added to a 25-ml calibrated flask.
(Table 6). The HPLC method, which was applied in another work for the determination of SDZ, trimetropim, and related compounds, was validated by means of external standards, using the standard Table 6 Direct determination tical preparations
of sulfadiazine
(in mg/tablet)
Pharm. preparation
Direct RTPF a
FIA-RTPF
Adiazine Triglobe
450 f 22 874 f 35
562 f 34 774 f 23
in pharmaceua
HPLC b 532+18 883 + 35
’ mg SDZ/tablet + standard deviation. b The HPLC instrument was a Perkin-Elmer Series 4, equipped with a constant-flow pump; together with a diode-array LC-235 detector and a laboratory computing integrator LCI-100. The wavelength of detection was 240 nm. The column was a Nucleosil 5 C-18 1OOA 25 X0.46 cm, from Phenomenex. Experimental conditions: a water (buffer: acetic acid, sodium acetate 0.01 M and phosphoric acid, sodium phosphate 0.05 M, pH 4.50)acetonitrile mobile phase, 83-17. Flow rate: 2 ml/min [21].
50
J.J. Aaron et al./Analytica
addition procedure [21]. As can be seen, the results evaluated by RTPF are close to those obtained by HPLC, except for the evaluation of an Adiazine preparation by FIA-RTPF. It shows the absence in RTPF of significative interference from the pharmaceutical matrix used.
Chimica Acta 314 (1995) 45-50
Governments, and the DGICYT of the Ministry of Education and Science of Spain (Project PB91-0856) for financial support of this work.
References 4. Conclusion
111J.W. Bridges, L.A. Gifford, W.P. Hayes, J.N. Miller and D. Thorburn,
We have demonstrated the importance of the role of a partially alcoholic medium to improve the performances of room-temperature photochemically induced fluorescence for determination of sulfadiazine, in terms of rapidity and precision. By reducing the optimal irradiation times needed in the methanolwater (5050, v/v> mixture used, it becomes possible to combine a RTPF detector with a FIA system, without detrimental consequences for the detection limit and the applicability of the method to pharmaceutical samples. The FIA procedure compares favourably to the bulk solution method in terms of rapidity, reliability and low absolute detection limits. Another advantage of FIA is that it could be easily automatized for SDZ routine analysis. We can also conclude from this work that we have improved the FL4 approach, relative to previous FIA systems used for the determination of other sulfonamides [15] and phenothiazines [18]; indeed, we have tested a longer photochemical reactor, and we have used a methanol-water (50:50, v/v) mixture, which results into an enhanced detectability of the analyte, and an increased rapidity of the FIA response.
Acknowledgements The authors acknowledge the Spanish-French Integrated Action (HF-41) of the Spanish and French
Anal. Chem., 46 (1974) 1010.
121T. Sakavo and T. Amano, Yakugaku Zasshi, 96 (1976) 1114. [31 J. Arthur F. de Silva and N. Strojny, Anal. Chem., 47 (1975) 714. 141 N. Takeda and Y. Akiyama, J. Chromatogr., 607 (1992) 31. [51 M.C. Mahedero, F. Salinas and J.J. Aaron, J. Pharm. Biomed. Anal., 12 (1994) 1097. 161T. Ahmad, Pharmazie, 37 (1982) 559. [71 S. Tammilehto, M. Lehtonen, 2nd International Symposium on Drug Analysis, Abstracts, Brussels, 1986, p. 241. 181 M.C. Mahedero and J.J. Aaron, Analusis, 20 (1992) 53. 191 M. Sanchez Pena, F. Salinas, M.C. Mahedero and J.J. Aaron, Talanta, 41 (1994) 233. HOI J.W. Birks and R.W. Frei, Trends Anal. Chem., 1 (1982) 361. [ill J.J. Aaron, in S.G. Schulman (Ed.), Molecular Luminescence Spectroscopy, Methods and Applications, Part 3, Wiley, New York, 1993, p. 85. WI M. Tsuchiya, E. Torres, J.J. Aaron and J.D. Winefordner, Anal. Len., 17 (1984) 1831. 1131 D. Chen, A. Rios, M.D. Luque de Castro and M. Valcarcel, Analyst, 116 (1991) 171. [141 D. Chen, A. Rios, M.D. Luque de Castro and M. Valcarcel, Talanta, 38 (1991) 1227. t151 M.C. Mahedero and J.J. Aaron, Anal. Chim. Acta, 269 (1992) 193. [I61 J. Martinez Calatayud and C. Gomez Benito, Anal. Chim. Acta, 256 (1992) 105. [171 A. Mellado Romero, C. Gomez Benito and J. Martinez Calatayud, Anal. Lett., 25 (1992) 1889. [181 B. Laassis, J.J. Aaron and M.C. Mahedero, Talanta, 41 (1994) 1985. [I91 N. Lammers, H. de Bree, C.P. Groen, H.M. Ruijten and B.J. de Jong, J. Chromatogr., 496 (1989) 291. Practice, University of DO1 R. Cela, Avarices en Quimiometria Santiago de Compostela, Publication and Scientific Exchange Service, Santiago de Compostela, Spain, 1994. ml M.C. Mahedero, personal communication.
Chimica
Acta 314 (1995) 45-50
Quantitative analysis of sulfadiazine using photochemically induced fluorescence detection in bulk solution and in a flow injection system * J.J. Aaron ay*, M.I. Acedo Valenzuela b, M. Sanchez Pena b, F. Salinas b, M.C. Mahedero b a Institut de Topologie et de Dynamique des Sys&mes de l’Universit6 Paris 7 - Denis Diderot, associk au CNRS URA 34, 1, rue Guy de la b Department ofAnalytical
Brosse, 75005 Paris, France Chemishy, Faculty ofSciences, University of Extramadura, 06071 Badajoz, Spain
Received 28 November
1994; revised 4 April 1995; accepted 9 May 1995
Abstract Room-temperature photochemically-induced fluorescence (RTF’F) methods, using bulk solution and flow-injection analysis (FIA) were proposed for determining sulfadiazine (SDZ). The RTPF determination was based on the irradiation of SDZ with ultraviolet light. A methanol-water (50:50, v/v) mixture was found to be the optimal solvent for the rapid, precise, and sensitive RTPF analysis of SDZ. Limits of detection were 40 ng/ml (bulk solution) and 90 ng/ml (FL4). The methods were applied for the determination of SDZ in pharmaceuticals. Satisfactory recoveries, ranging between 93 and 111.8%, were obtained. Keywords: Fluorimetry; Flow injection; Sulfadiazine
1. Introduction Due to their increasing importance in medicine and veterinary practice, sulfonamides have been the subject of a number of analytical studies. The fluo-
* Presented, in part, at the VIth International Symposium on Luminescence Spectroscopy in Biomedical Analysis - Detection Techniques and Applications in Chromatography and Capillary Electrophoresis, Bruges, Belgium, June 5- 7, 1994. The majority of papers presented at this symposium have been published in Anal. Chim. Acta, Vol. 303, No. 1. Corresponding author. l
0003-2670/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0003-2670(95)00254-S
rescence of several sulfonamides has been investigated, and some fluorimetric methods have recently been proposed as a detection means in high-performance liquid chromatography (HPLC) D-51. However, the use of these techniques has been limited in the case of nitrogen heterocyclic sulfonamides, which generally present a weak fluorescence. Studies on the photochemical decomposition of sulfonamides were reported a few years ago [6,7]. More recently, it was shown by us that room-temperature photochemically induced fluorescence (RTPF) of several heterocyclic sulfonamides could be used to determine these compounds at the ppb level in
46
J.J. Aaron et al./Analytica
aqueous solution [8]. Ultraviolet (UV) irradiation times required for obtaining maximum fluorescence signals of the photoproducts were between 10 and 30 min. These irradiation times can be significantly reduced by utilizing non-aqueous alcoholic media
[91. On the other hand, photochemical fluorescence detectors have been mainly applied to HPLC [lO,ll], but they have been used less frequently for improving the fluorescence detection of analytes in simple hydrodynamic systems such as flow-injection analysis (FIA) [12-181. For example, Mahedero and Aaron have proposed a FIA-RTPF method for determination of sulfamethazine [15]. Sulfadiazine (SDZ), is an heterocyclic sulfonamide which is currently used in the treatment of urine infections and for ophthalmology applications. Several methods such as HPLC [19], and RTPF in aqueous medium [8], have been proposed for its determination. In this paper, we investigated the effects of alcohols and alcohol-water mixtures on the photochemitally induced fluorescence intensity of sulfadiazine. The proposed RTPF methods, using bulk solution and FIA have been applied to the determination of sulfadiazine in pharmaceutical preparations, and the results have been compared with those obtained by HPLC.
2. Experimental 2.1. Apparatus A Kontron Model SFM-25 spectrofluorometer interfaced with a Geocom microcomputer was used for fluorescence measurements in both bulk solution and FIA methods. An Osram 200 W high-pressure mercury arc lamp with an Oriel Model 8500 power supply was used for photochemical studies in bulk solution. The FIA-photochemical fluorescence system has been described previously [15]. 2.2. Reagents Sulfadiazine (SDZ) was purchased from Sigma. Pharmaceutical preparations of sulfadiazine were a generous gift of Lab. Doms-Adrian, (Courbevoie,
Chimica Acta 314 (1995) 45-50
France; Adiazine, composition per tablet: 500 mg sulfadiazine, excipient, q.s.1 and of Lab. Astra-Ifesa (Barcelona, Spain; Triglobe, composition per tablet: 180 mg trimetoprim, 820 mg sulfadiazine, excipient q.s.). Standard stock solutions of SDZ (250 pg ml-‘) were prepared by dissolving the compound in methanol (Merck, spectroscopic grade). All SDZ samples were prepared taking convenient aliquots from standard solutions and diluted to the mark in a calibrated flask with water, methanol, ethanol (Merck, spectroscopic grade), 2-propanol (Merck, analytical grade) or methanol-water mixtures. The methanol-water mixtures were prepared just before use at room temperature to avoid contraction of volume and heat releasing. 2.3. Procedure For the bulk solution method, an aliquot of the SDZ sample containing between 0.1 and 4.1 pg ml-l in a methanol-water (50:50, v/v) mixture was placed in a l-cm quartz cuvette and irradiated at room temperature with the high-pressure mercury lamp for 2.5 min. Fluorescence intensity measurements were performed at the excitation and emission analytical wavelengths of 295 and 352 nm, respectively. Linear calibration graphs were established with the SDZ solutions of known concentrations treated in the same conditions as the pharmaceutical samples. Triplicate measurements were done in all cases. In the case of the FIA method [15] a water carrier, a 4.2-ml min-’ flow rate, a 300-~1 injected volume, and a 250-cm photoreactor length were used. Samples containing between 0.2 and 6.5 pg ml-’ of SDZ in methanol-water (50:50, v/v) were injected. No cleaning up procedure was used prior to FIA analysis. Fluorescence intensity measurements were obtained with excitation and emission analytical wavelengths of 295 and 352 nm, respectively. Linear calibration graphs were established with SDZ solutions of known concentrations treated in the same way as the pharmaceutical samples. All measurements were performed in triplicate. For the applications to Adiazine and Triglobe pharmaceutical preparations, three tablets of each compound were measured by weight and powdered. The solutions of these drugs were prepared by dis-
J.J. Aaron et al. /Analytica
solving 25.9 and 11.1 mg of each compound, tively, in a lOO-ml volume of methanol.
respec-
47
Chimica Acta 314 (1995) 45-50 Table 1 Effect of alcohols on the photochemically properties of sulfadiazine
induced
fluorescence
3. Results and discussion
Previously, we used an optimal UV irradiation time of 18 min for determining SDZ in aqueous solution by RTPF [8]. In order to examine if it was possible to reduce significantly this irradiation time to maximum fluorescence value - corresponding signal of the photoproducts - we investigated the effect of alcoholic solvents on the photochemically induced fluorescence properties of SDZ. The alcohols assayed were methanol, ethanol, and 2-propanol. Fig. 1 shows the effect of W irradiation time on the fluorescence intensity of SDZ at maximum emission wavelength in different alcoholic media. All curves indicate an important increase of fluorescence signal with time. In all instances, the excitation wavelength was fixed at 295 nm. The fluorescence properties of SDZ in the various solvents under study are compared in Table 1. A slight red shift of the SDZ emission wavelength was noted upon going from methanol to ethanol and 2-propanol, and from
a Optimal irradiation Fence intensity. Maximum relative concentration of SDZ where it was 1.04 pg
0
1 .o
2.0
Tirr
3.0
4.0
(min)
Fig. 1. Effect of the ultraviolet irradiation intensity of SDZ in various alcohols.
time on the fluorescence
420 108 115 -
time corresponding
to the maximum
fluores-
fluorescence intensity. In all instances, the was 2.08 pg ml-‘, except in water medium ml-‘.
the alcohols to water. Photochemically induced fluorescence intensity maxima (I,,,) were larger in aqueous medium than in alcohols. Also, it can be seen that the optimal irradiation time ranges correwere much shorter in alcoholic sponding to I,,,, solvents (0.5-4 min) than in water (8-18 min) (see Table 1 and Fig. 1). In order to improve the rapidity and sensitivity of the RTPF method, it is desirable to use shorter irradiation times and to obtain a larger fluorescence signal. Therefore, we decided to investigate the effect of varying the alcohol percentage of
01 5.0
8-18 OS-l.5 l-2 1.5-4
361 345 350 354
Water Ethanol Methanol 2-propanol
3.1. Effect of the alcohols
I
,
1
,
I
,
I
,
2
Tir’r (mi;)
’
Fig. 2. Effect of the ultraviolet time on the fluorescence intensity of SDZ in methanol:water mixtures with various methanol percentages.
48
J.J. Aaron et al. /Analytica
Table 2 Effect of the methanol fluorescence properties
Chimica Acta 314 (1995) 45-50
3.2. Determination percentage of SD2
% methanol ’
on the photochemical b ?iin,
I max c
2-3 2.2-3.5 4.5-6.5
280 530 420
;;) 347 352 354
70 SO 30
a Percentage of methanol (v/v) in the methanol-water mixture. b Optimal irradiation time range. ’ Maximum relative fluorescence intensity. In all instances, the concentration of SD2 was 1.04 J.J~ ml-‘.
alcohol-water mixtures on these fluorescence parameters. For this study, methanol was chosen since it provided an irradiation time smaller and a fluorescence signal larger than other alcohols. Fig. 2 and Table 2 show that an increase of methanol percentage from 30 to 70% produces a marked decrease of irradiation time, while the fluorescence intensity maximum is larger for a methanol-water (5050, v/v) mixture. The larger fluorescence signal found in the latter medium compared to 30% and 70% methanol media can be attributed to a specific and enhanced solvation of the SDZ photoproduct singlet excited state in this particular mixture. Therefore, the methanol-water (50:50, v/v) medium was considered as optimal for the RTPF determination of SDZ in bulk solution. Moreover, an optimum irradiation time value of 2.5 min was selected for all analytical measurements.
Table 3 Analytical
figures of merit for the RTPF determination
Method
Concentration
range
Regression
of sulfadiazine
in bulk solutions
induced
of sulfadiazine equation b
( pg/mll
A
B
Bulk solution
0.1-4.1
FIA
0.2-6.5
26.5 (4.7) = 26.8 (4.3) =
234.7 (2.2) c 84.3 (1.3) c
The analytical figures of merit for the determination of SDZ in methanol-water (50:50, v/v) are given in Table 3. A linear calibration plot was obtained between SDZ concentrations of 0.1 and 4.1 pg/ml. The correlation coefficient was close to unity. The limit of detection (LOD) value was rather low (40 ng/ml), although it was higher than that obtained previously for the determination of SDZ in water 181. The relative standard deviation (RSD) value was particularly small (1.7% for a SDZ concentration of 2.6 pg/ml), indicating an excellent reproducibility of RTPF measurements in bulk solutions. 3.3. Determination
of suljadiazine
in a FIA system
The influence of FL4 parameters on the RTPF signal of SDZ was investigated. The flow-rate, photoreactor length, and injected volume were optimized to provide maximum RTPF response and minimum band broadening, as in our previous studies [15,18]. The optimal values for these three FIA parameters were found to be 4.2 ml/min, 250 cm and 300 ~1, respectively. Table 3 summarizes the analytical figures of merit. A linear calibration graph was established over more than one order of magnitude, with a correlation coefficient of 0.999. The LOD value (90 ng/ml) is rather low, corresponding to an absolute detectable amount of 27 ng. The RSD value was rather small (2.1% for a SDZ concentration of 2.1
in a 50% (v/v> water-methanol Residual standard deviation .Sy/x
mixture a
LOD d
LOQ ’
RSD *,s
(rig/ml)
(ng/ml)
(%o)
14.3
40
150
12.7
90
310
1.7 5.2 2.1 4.5
* s * s
A, = 295 mm; A,, = 352 mm; tirr = 2.5 min. Ref. 1201 was used for the calculation of statistical data. I, = A + B . c. IF = relative fluorescence intensity; c = analyte concentration ( pg/ml). Correlation coefficient = 0.999 in both cases. Estimated standard deviation of A and B values. LOD = limit of detection, defined as the concentration of analyte giving a signal-to-noise ratio of 3. LOQ = limit of quantification, defined as the concentration of analyte giving a signal-to-noise ratio of 10. RSD = relative standard deviation for a SDZ concentration of 2.6 pg/ml (bulk solution) and 3.1 pg/ml (FL41 (n = 10). RSD for a SDZ concentration of 0.5 pg/ml (n = 10).
J.J. Aaron et al. /Analytica Table 4 Determination tion method)
of SDZ in pharmaceutical
Adiazine * added (ml) 0.3 0.3 0.3 0.3 0.3 0.2 0.2 0.2 0.2 0.2 Triglobe b added (ml) 0.4 0.4 0.4 0.4 0.4 0.6 0.6 0.6 0.6 0.6
preparations
(bulk solu-
Table 5 Determination method)
of
SDZ
49
in
pharmaceutical
preparations
SDZ added
SDZ found
Recovery
SDZ added
SDZ found
Recovery
(ppm)
(ppm)
(o/o)
(ppm)
(ppm)
(%I
-
0.64 2.48 3.03 3.54 1.76 2.81 3.24 3.63 4.68
-
2.00 2.57 3.31 3.64 4.01 1.43 1.91 2.63 3.07 3.46
0.54 1.07 1.61 2.15 0.54 1.07 1.72 2.15
-
1.63 2.21 2.87 3.37 3.76 2.42 2.71 3.02 3.40 3.59
0.52 1.03 1.55 2.06 0.31 0.69 0.93 1.24
Adiazine a added (ml) 0.1 0.1 0.1 0.1 0.2 0.2 0.2 0.2 0.2 Triglobe b added (ml) 0.4 0.4 0.4 0.4 0.6 0.6 0.6 0.6
101.2 107.8 100.8 96.6 96.9 105.2 97.4 96.6
_ 103.0 107.8 106.0 101.8 99.3 97.1 101.6 98.1
a Adiazine solution prepared dissolving 25.9 mg of pharmaceutical preparation in 100 ml of methanol. Milliliters added to a 25-ml calibrated flask. b Triglobe solution prepared dissolving 11.1 mg of pharmaceutical preparation in 100 ml of methanol. Milliliters added to a 25-ml calibrated flask.
pg/ml), showing the FIA method. 3.4. Analytical
Chimica Acta 314 (1995) 45-50
a satisfactory
reproducibility
of
applications
The proposed RTPF methods were applied for the determination of SDZ in pharmaceutical preparations. In all cases, the standard addition procedure was used. Typical results are shown in Tables 4 and 5, for bulk solution and for FIA methods, respectively. Satisfactory recovery values were obtained, ranging from 96.6 to 107.8% for bulk solution methods, and from 92.9 to 111.8% for the FIA method. The absolute weights of SDZ contained in the pharmaceutical preparations were measured directly by both methods, and validated by the HPLC method
1.58 2.11 2.63 1.05 1.58 2.14 2.63
-
111.8 110.1 108.2 _ 99.8 96.8 92.9 106.4
-
1.50 2.40 3.07 3.50 2.38 2.71 2.96 3.29
1.03 1.55 2.06 _ 0.31 0.62 0.93
(FIA
94.9 100.5 98.1 100.4 98.6 99.4
a Adiazine solution prepared dissolving 25.9 mg of pharmaceutical preparation in 100 ml of methanol. Milliliters added to a 25-ml calibrated flask. b Triglobe solution prepared dissolving 11.1 mg of pharmaceutical preparation in 100 ml of methanol. Milliliters added to a 25-ml calibrated flask.
(Table 6). The HPLC method, which was applied in another work for the determination of SDZ, trimetropim, and related compounds, was validated by means of external standards, using the standard Table 6 Direct determination tical preparations
of sulfadiazine
(in mg/tablet)
Pharm. preparation
Direct RTPF a
FIA-RTPF
Adiazine Triglobe
450 f 22 874 f 35
562 f 34 774 f 23
in pharmaceua
HPLC b 532+18 883 + 35
’ mg SDZ/tablet + standard deviation. b The HPLC instrument was a Perkin-Elmer Series 4, equipped with a constant-flow pump; together with a diode-array LC-235 detector and a laboratory computing integrator LCI-100. The wavelength of detection was 240 nm. The column was a Nucleosil 5 C-18 1OOA 25 X0.46 cm, from Phenomenex. Experimental conditions: a water (buffer: acetic acid, sodium acetate 0.01 M and phosphoric acid, sodium phosphate 0.05 M, pH 4.50)acetonitrile mobile phase, 83-17. Flow rate: 2 ml/min [21].
50
J.J. Aaron et al./Analytica
addition procedure [21]. As can be seen, the results evaluated by RTPF are close to those obtained by HPLC, except for the evaluation of an Adiazine preparation by FIA-RTPF. It shows the absence in RTPF of significative interference from the pharmaceutical matrix used.
Chimica Acta 314 (1995) 45-50
Governments, and the DGICYT of the Ministry of Education and Science of Spain (Project PB91-0856) for financial support of this work.
References 4. Conclusion
111J.W. Bridges, L.A. Gifford, W.P. Hayes, J.N. Miller and D. Thorburn,
We have demonstrated the importance of the role of a partially alcoholic medium to improve the performances of room-temperature photochemically induced fluorescence for determination of sulfadiazine, in terms of rapidity and precision. By reducing the optimal irradiation times needed in the methanolwater (5050, v/v> mixture used, it becomes possible to combine a RTPF detector with a FIA system, without detrimental consequences for the detection limit and the applicability of the method to pharmaceutical samples. The FIA procedure compares favourably to the bulk solution method in terms of rapidity, reliability and low absolute detection limits. Another advantage of FIA is that it could be easily automatized for SDZ routine analysis. We can also conclude from this work that we have improved the FL4 approach, relative to previous FIA systems used for the determination of other sulfonamides [15] and phenothiazines [18]; indeed, we have tested a longer photochemical reactor, and we have used a methanol-water (50:50, v/v) mixture, which results into an enhanced detectability of the analyte, and an increased rapidity of the FIA response.
Acknowledgements The authors acknowledge the Spanish-French Integrated Action (HF-41) of the Spanish and French
Anal. Chem., 46 (1974) 1010.
121T. Sakavo and T. Amano, Yakugaku Zasshi, 96 (1976) 1114. [31 J. Arthur F. de Silva and N. Strojny, Anal. Chem., 47 (1975) 714. 141 N. Takeda and Y. Akiyama, J. Chromatogr., 607 (1992) 31. [51 M.C. Mahedero, F. Salinas and J.J. Aaron, J. Pharm. Biomed. Anal., 12 (1994) 1097. 161T. Ahmad, Pharmazie, 37 (1982) 559. [71 S. Tammilehto, M. Lehtonen, 2nd International Symposium on Drug Analysis, Abstracts, Brussels, 1986, p. 241. 181 M.C. Mahedero and J.J. Aaron, Analusis, 20 (1992) 53. 191 M. Sanchez Pena, F. Salinas, M.C. Mahedero and J.J. Aaron, Talanta, 41 (1994) 233. HOI J.W. Birks and R.W. Frei, Trends Anal. Chem., 1 (1982) 361. [ill J.J. Aaron, in S.G. Schulman (Ed.), Molecular Luminescence Spectroscopy, Methods and Applications, Part 3, Wiley, New York, 1993, p. 85. WI M. Tsuchiya, E. Torres, J.J. Aaron and J.D. Winefordner, Anal. Len., 17 (1984) 1831. 1131 D. Chen, A. Rios, M.D. Luque de Castro and M. Valcarcel, Analyst, 116 (1991) 171. [141 D. Chen, A. Rios, M.D. Luque de Castro and M. Valcarcel, Talanta, 38 (1991) 1227. t151 M.C. Mahedero and J.J. Aaron, Anal. Chim. Acta, 269 (1992) 193. [I61 J. Martinez Calatayud and C. Gomez Benito, Anal. Chim. Acta, 256 (1992) 105. [171 A. Mellado Romero, C. Gomez Benito and J. Martinez Calatayud, Anal. Lett., 25 (1992) 1889. [181 B. Laassis, J.J. Aaron and M.C. Mahedero, Talanta, 41 (1994) 1985. [I91 N. Lammers, H. de Bree, C.P. Groen, H.M. Ruijten and B.J. de Jong, J. Chromatogr., 496 (1989) 291. Practice, University of DO1 R. Cela, Avarices en Quimiometria Santiago de Compostela, Publication and Scientific Exchange Service, Santiago de Compostela, Spain, 1994. ml M.C. Mahedero, personal communication.