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Study on fluorescent property of degrading products of piperacillin and its analytical application
Spectrochimica Acta Part A 56 (2001) 217 – 222 www.elsevier.nl/locate/saa
Study on fluorescent property of degrading products of piperacillin and its analytical application Bo Tang *, Li Ma, Yue Sun, Huai-you Wang Department of Chemistry, Shandong Normal Uni6ersity, Jinan, 250014, Peoples Republic of China Received 31 March 2000; accepted 31 May 2000
Abstract The fluorimetric property of the degrading products of piperacillin has been studied in detail. The studies on degrading pH, degrading time, detection alkalinity and other corresponding analytical parameters of acid degradation have been made. Then fluorometry of piperacillin was established by producing its stable fluorescent products. The detection limit for acid degradation analytical method is 2.34 ng/ml, the linear range is 7.80 – 4.0×102 ppb. The analytical sensitivity, precision and stability of degrading products of acid degradation are satisfactory, which has been used for the determination of the trace piperacillin in human serum and urine with satisfactory results. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Antibiotic; Sodium piperacillin; Degradation; Fluorescence analysis; Human serum and urine
1. Introduction Penicillin is a kind of important b-lactam antibiotic, which can bind with the penicillin-binding protein (PBP) on the cellular membrane, interrupt the synthesis of viscous phthalein of bacteria cellular wall and make it fail to cross link, which leads to cellular wall’s coloboma. Therefore, bacteria will die from cellular wall’s disruption. Piperacillin (PIPC, the structure is shown in Scheme 1) is a relatively new semisynthetic penicillin with a broad spectrum of activity to both gram-negative and gram-positive anaerobic and
* Corresponding author.
aerobic organisms [1]. Piperacillin was shown to have considerable antibacterial activity against a wide range of bacterial pathogens, such as colibacillus, pyocyanine, mycetozoan, pneumobacillus and salmonella. The activities of PIPC was found to be more efficient than many other penicillins, such as ticarcillin, carbenicillin, ampicillin and cephalosporins [2–6]. Piperacillin has also a very high affinity for penicillin-binding proteins [7]. It is rather suitable to serious infection of gram-negative bacilli and the impair of kidney function. Piperacillin has little toxicity and sideeffect. After injection, there is a relatively high blood concentration. Therefore, it has a widespread clinical application [8]. Methods to determine PIPC have been published in the literature, such as chromatography
1386-1425/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 1 4 2 5 ( 0 0 ) 0 0 3 4 7 - 4
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B. Tang et al. / Spectrochimica Acta Part A 57 (2001) 217–222
[9–12], microbiological method [13 – 15], mercurimetric determination [16,17] and electrochemistry [18–21]. Among them high performance liquid chromatography (HPLC) [22 – 31] is mostly used. But by now fluorescence analytical method has not been reported. It is an advanced subject in analytical chemistry to use highly sensitive fluorescence analysis to study and determine the pharmaceuticals’ present quantities and configurations in endomic parts and excrements [32]. The method that determine antibiotic by measuring the fluorescence intensity of its degrading products is simple, rapid and has a good sensitivity, precision and recovery [33,34]. This method can be applied to the clinic pharmacology study of piperacillin. We found that piperacillin could degrade in acid, alkali or neutral acquous solution, particularly remarkable when the PH value was below 4.0 or above 7.0, and that the fluorometry with acid degradation had more advantages of analytical precision and was more accurate than that with alkali degradation. The fluorescence analysis for acid degrading products has been made for the first time in this paper. In terms of the stability of degrading products and the detection limit, acid degradation is suitable to fluorescence analysis. The fluorescence intensity of degrading products was affected by a lot of factors and this paper made a deep study of them.
2. Experimental
2.1. Apparatus All fluorescence measurements were carried out on a Perkin – Elmer (Norwalk, CT, USA) LS-5 spectrofluorimeter, equipped with a xenon lamp, 1.0 cm quartz cells and a Perkin – Elmer Model
Scheme 1.
561 recorder. All pH measurements were made with a PH-3C digital pH meter (Shanghai Lei Ci Device Works, Shanghai, China) with a combined glass-Calomel electrode.
2.2. Reagents Piperacillin was of analytical reagent grade and purchased from Beijing Chemical Corporation, China. Other chemicals used were of analytical reagent grade. Distilled deionized water was used through out the experiment. The standard stock solution (1 mg/ml) was prepared by dissolving 0.1000 g sodium piperacillin (accurately weighed) with distilled deionized water and dilute it to volume in a 100 ml volumetric flask. The stock solution was protected from light and kept in a refrigerator. The working solution (0.5 mg/ml) were prepared by dilution.
2.3. Procedure Into a 10 ml colorimetric tube were added a certain amount of sodium piperacillin stock solution, 1.5 ml of 2.0 mol/l H2SO4 sequentially. The tube was then diluted to the 5 ml mark with distilled deionized water. The solution was mixed and then degraded in a boiling water-bath for 130 min. After cooling under running water to the room temperature immediately, add 2 mol/l NaOH to adjust the pH value to 7. Another 1.5 ml of 2 mol/l NaOH was added and it was diluted to volume. After shaking, the fluorescent intensity of the solution was measured at 424 nm with excitation at 336 nm against a reagent blank.
2.4. Samples treatment Into a 20 ml centrifuge tube were added 100 mg sodium piperacillin standard solution, 2.5 ml human serum and 5.0 ml 10% trichloroacetic acid in succession, then dilute it with distilled deionized water to the mark. Centrifugalize it for 10 min (3000 rpm). The supernatant was transfered into a separating funnel and extracted three times with
B. Tang et al. / Spectrochimica Acta Part A 57 (2001) 217–222
219
3. Result and discussion
3.1. Excitation and emission spectra
Fig. 1. (a) Excitation and (b) emission spectra of degrading products of piperacillin. Peaks: 1 and 1%, degrading products of piperacillin. 2 and 2%, reagent blank. CPIPC = 0.5 mg/mol; degrading time: 130 min; detection alkalinity: [OH−] =0.3 mol/l; degrading acidity: [H2SO4] = 0.3 mol/l.
In order to determine the optimum working wavelength, the spectral characteristics of degrading products of piperacillin were studied. The corrected excitation and emission spectra (Fig. 1) showed that the wavelengths of maximum excitation and emission of degrading products of PIPC were 336 and 424 nm, respectively.
3.2. Optimization of experimental 6ariables The experimental variables were optimized by applying the univariate method. Degrading time has an important effect on the fluorescence intensity. As can be seen in Fig. 2, the fluorescence intensity varied significantly with changes in degrading time. At the beginning of the reaction, the fluorescence intensity of products increased with the increase of degrading time up to 110 min, then remained constant between 120 and 130 min and decreased thereafter. So, a 130 min degrading time was adopted. The fluorescence intensity of acid degrading products varied with changes in the concentration of H2SO4. Fig. 3 showed that the fluorescence intensity was relatively high and almost remained
Fig. 2. The relationship between relative fluorescence intensity and degrading time. Degrading acidity: [H2SO4] =0.3 mol/l; detection alkalinity: [OH−] = 0.3 mol/l; CPIPC = 0.5 mg/mol.
10 ml portions of ether. Transfer the aqueous layer to a 25 ml volumetric flask and dilute it to the mark.After shaking, keep it in a refrigerator for further use. Divide 10 ml urine of adult men into two portions and add them to 25 ml volumetric flasks, respectively. Into one portion was added 100 mg sodium piperacillin and use the other as a reagent blank. Then dilute them to the mark. After shaking, they were protected from light and kept in a refrigerator.
Fig. 3. The relationship between relative fluorescence intensity and degrading acidity. CPIPC =0.5 mg/mol; detection alkalinity: [OH−]= 0.3 mol/l; degrading time: 130 min.
B. Tang et al. / Spectrochimica Acta Part A 57 (2001) 217–222
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3.3. The stability of the fluorescence intensity of degrading products
Fig. 4. The relationship between relative fluorescence intensity and detection alkalinity. CPIPC = 0.5 mg/mol; degrading time: 130 min; degrading acidity: [H2SO4] =0.3 mol/l.
The stability of the fluorescence intensity of degrading products had an immediate influence on the sensitivity and accuracy. Under the established experimental conditions, make the acid degrading products of 0.5 mg/ml sodium piperacillin exposed to light in light path for various periods of time and then determine the fluorescence intensity. The results are shown in Table 1. From Table 1 it can be seen that within 120 min, the relative deviation of fluorescence measurements was less than 9 2%, which indicated that the degrading products were rather constant. We supposed, with the excitation of strong ultraviolet radiation, initially there was an intramolecular phototautomerism and then partial photolysis happened. Because the ultraviolet intensity indoors was very weak, we ignored the small change of fluorescence intensity during the course of determination. So, fluorometric determination was adaptable to determine degradation products.
3.4. Analytical characteristics constant over the concentration of H2SO4 range 0.2–0.4 mol/l and decreased rapidly outside this concentration range. Therefore, a concentration of H2SO4 of 0.3 mol/l was recommended for use. The influence of detection alkalinity on the fluorescence intensity was also studied under the established experimental conditions. As can be seen in Fig. 4, the fluorescence intensity reached a maximum over the [OH−] range 0.25 – 0.35 mol/l, outside of which smaller intensity was observed. So, 0.3 mol/l was chosen as the detection alkalinity.
Under the optimum experimental conditions, there was a linear relationship between the fluorescence intensity and the concentration of PIPC in the range 7.80–400 ppb with a correlation coefficient (r) of 0.9958. The linear regression equation was DIF = 108.70CPIPC (mg/ml) −1.337. The standard deviation of the fluorescence measurements was 0.085 obtained from a series of 11 blank solutions. The limits of detection (k =3) and of determination (k= 10) of the method were established according to the IUPAC definitions (C1 = KS0/S,where C1 is the limit of detection, S0
Table 1 The quenching of degrading products’ fluorescence Lighting time (min) Fluorescence intensitya RD% a
0 53.00
10 20 30 40 50 60 70 80 90 100 110 52.33 52.63 52.55 52.70 52.93 52.55 52.48 52.63 52.30 52.26 52.30 −1.26 −0.70 −0.84 −0.56 −0.14 −0-84 −0.98 −0.70 −1.26 −1.39 −1.26
Mean value of six measurements.
120 52.40 −1.12
B. Tang et al. / Spectrochimica Acta Part A 57 (2001) 217–222 Table 2 Determination of PIPC in human urine and serum samples (P= 0.95) Samples
Serum Urine a
PIPC added (mg/ml)
0.100 0.100
a
Found (mg/ml) Proposed method
HPLC method [22]
0.09790.004 0.1019 0.001
0.1029 0.003 0.0989 0.005
Each sample was analyzed six times.
the standard error of blank determination, S the slope of the standard curve and K is the constant related to the confidence interval) [35] and the values found were 2.34 and 7.80 ppb, respectively. The relative standard deviation was 2.54%, obtained from a series of ten standards each containing 250 ppb of piperacillin.
3.5. Sample analysis Into a 10 ml colorimetric tube was added 2.5 ml human serum or urine that had been treated.The procedure was used to the determination of trace PIPC in them. The results are given in Table 2.
4. Conclusions
.
From the results presented it can be concluded that the proposed method for the determination of trace PIPC is simple, rapid, stable, sensitive and inexpensive.
Acknowledgements This work was supported by the Natural Science Foundation of China (no. 29975016).
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References [1] L. Verbist, Antimicrob. Ag. Chemother. 13 (1978) 249. [2] A.L. Barry, Cleveland Clin. Quart. 47 (1980) 311. [3] D.A. Leigh, K.T. Simmons, Drugs Exp. Clin. Res. 5 (1979) 99. [4] N.A. Kuck, G.S. Redin, J. Antibiot. 31 (1978) 1175. [5] R.J. Fass, J. Barnishan, Rev. Infect. Dis. 2 (1980) 841. [6] R.J. Fass, Antimicrob. Ag. Chemother. 21 (1982) 1003. [7] G.A. Botta, J.T. Park, J. Bact. 145 (1981) 333. [8] Y.Y. Zhang et al., Antibiotic (China) 7 (6) (1982) 383– 391. [9] Z. Plotkowiak, Z. Seyda, Farm. Pol. 48 (8) (1992) 493– 497. [10] G. Peytavin, A. Baillet, R. Farinotti, J. Pharm. Clin. 10 (1) (1991) 9 – 19. [11] H. Nishi, T. Fukuyama, M. Matsuo, J. Chromatogr. 515 (1990) 245 – 55. [12] M. Park, Y.H. Cho, J. Yang, Yakhak Hoechi, 28 (1) (1984) 25 – 7. [13] R. Wang, X. Sun, Chin. J. Pharm. Anal. 11 (5) (1991) 282 – 284. [14] S. Kumiko, S. Toru et al., Chemotherapy (Tokyo) 42 (Suppl. 2) (1994) 263 – 76. [15] W. Cullmann, W. Dick, M. Edelmann, J. Clin. Chem. Clin. Biochem. 23(3) (1985) 151 – 6. [16] B. Pospisilova, M. Simkova, Kubes, J. Pharmazie. 41 (10) (1986) 705 – 8. [17] B. Pospisilova, M. Simkova, Kubes, J. Cesk. Farm. 34 (3 – 4) 1085. [18] N. Abo El-Maali, M.A. Ghandour, M. Khodari, Talanta, 40 (12) (1993) 1833 – 1838. [19] L. Campanella, F. Mazzei et al., Ann. Chim. (Rome) 80 (9 – 10) (1990) 395 – 412. [20] L. Campanella, F. Mazzei et al., J. Pharm. Biomed. Anal. 6 (3) (1988) 299 – 305. [21] S. Schroeder, R. Voigt et al., Pharmazie. 40 (5) (1985) 333 – 5. [22] J.C. Garcia-Glez, R. Mendez et al., J. Chromatogr. A 812 (1 – 2) (1998) 213 – 220. [23] M.A. Campanero, A.M. Zamarreno et al. Chromatographia 46 (7 – 8) (1997) 374 – 380. [24] T. Tsukamoto, T. Ushio, J. Chromatogr. A 678 (1) (1994) 69 – 76. [25] S.L. Qiu, J.H. Liu, Chin. J. Pharm. Anal. 9 (1) (1989) 27 – 9, 17. [26] V. Gautier, F. Demotes-Mainard, J. Pharm. Biomed. Anal. 9 (2) (1991) 183 – 186. [27] A.P. Ocampo, K.O. Hoyt et al., J. Chromatogr. 496 (1989) 167. [28] C.E. Parker, J.R. Perkins et al., J. Chromatogr., Biomed. Appl. 127 (1993) 45 – 57. [29] A. Zarzuelo, F.G. Lopez et al., J. Liq. Chromatogr. Relat. Technol. 19 (4) (1996) 601 – 610. [30] K. Iwaki, N. Okumura et al. J. Chromatogr. 504(2) (1990) 359 – 67.
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[31] T. Marunaka, M. Maniwa et al., J. Chromatogr., Biomed. Appl. 75 (1988) 87–101. [32] D.K. An, Pharmaceutical Analysis, second ed, Beijing Press of People Hygiene, 1991, p. 334. [33] B. Tang, X.W. He, H.X. Shen, Chin. J. Anal. Chem. 22 (11) (1994) 1089.
.
[34] X.W. He, B. Tang, H.X. Shen, Chem. J. Chin. Uni. 16 (1) (1995) 26. [35] H.M.N.H. Irving, H. Freiser, T.S. West, (Eds.) IUPAC Compendium of Analytical Nomenclature, Definitive Rules, Pergamon Press, Oxford, 1981.
Study on fluorescent property of degrading products of piperacillin and its analytical application Bo Tang *, Li Ma, Yue Sun, Huai-you Wang Department of Chemistry, Shandong Normal Uni6ersity, Jinan, 250014, Peoples Republic of China Received 31 March 2000; accepted 31 May 2000
Abstract The fluorimetric property of the degrading products of piperacillin has been studied in detail. The studies on degrading pH, degrading time, detection alkalinity and other corresponding analytical parameters of acid degradation have been made. Then fluorometry of piperacillin was established by producing its stable fluorescent products. The detection limit for acid degradation analytical method is 2.34 ng/ml, the linear range is 7.80 – 4.0×102 ppb. The analytical sensitivity, precision and stability of degrading products of acid degradation are satisfactory, which has been used for the determination of the trace piperacillin in human serum and urine with satisfactory results. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Antibiotic; Sodium piperacillin; Degradation; Fluorescence analysis; Human serum and urine
1. Introduction Penicillin is a kind of important b-lactam antibiotic, which can bind with the penicillin-binding protein (PBP) on the cellular membrane, interrupt the synthesis of viscous phthalein of bacteria cellular wall and make it fail to cross link, which leads to cellular wall’s coloboma. Therefore, bacteria will die from cellular wall’s disruption. Piperacillin (PIPC, the structure is shown in Scheme 1) is a relatively new semisynthetic penicillin with a broad spectrum of activity to both gram-negative and gram-positive anaerobic and
* Corresponding author.
aerobic organisms [1]. Piperacillin was shown to have considerable antibacterial activity against a wide range of bacterial pathogens, such as colibacillus, pyocyanine, mycetozoan, pneumobacillus and salmonella. The activities of PIPC was found to be more efficient than many other penicillins, such as ticarcillin, carbenicillin, ampicillin and cephalosporins [2–6]. Piperacillin has also a very high affinity for penicillin-binding proteins [7]. It is rather suitable to serious infection of gram-negative bacilli and the impair of kidney function. Piperacillin has little toxicity and sideeffect. After injection, there is a relatively high blood concentration. Therefore, it has a widespread clinical application [8]. Methods to determine PIPC have been published in the literature, such as chromatography
1386-1425/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 1 4 2 5 ( 0 0 ) 0 0 3 4 7 - 4
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B. Tang et al. / Spectrochimica Acta Part A 57 (2001) 217–222
[9–12], microbiological method [13 – 15], mercurimetric determination [16,17] and electrochemistry [18–21]. Among them high performance liquid chromatography (HPLC) [22 – 31] is mostly used. But by now fluorescence analytical method has not been reported. It is an advanced subject in analytical chemistry to use highly sensitive fluorescence analysis to study and determine the pharmaceuticals’ present quantities and configurations in endomic parts and excrements [32]. The method that determine antibiotic by measuring the fluorescence intensity of its degrading products is simple, rapid and has a good sensitivity, precision and recovery [33,34]. This method can be applied to the clinic pharmacology study of piperacillin. We found that piperacillin could degrade in acid, alkali or neutral acquous solution, particularly remarkable when the PH value was below 4.0 or above 7.0, and that the fluorometry with acid degradation had more advantages of analytical precision and was more accurate than that with alkali degradation. The fluorescence analysis for acid degrading products has been made for the first time in this paper. In terms of the stability of degrading products and the detection limit, acid degradation is suitable to fluorescence analysis. The fluorescence intensity of degrading products was affected by a lot of factors and this paper made a deep study of them.
2. Experimental
2.1. Apparatus All fluorescence measurements were carried out on a Perkin – Elmer (Norwalk, CT, USA) LS-5 spectrofluorimeter, equipped with a xenon lamp, 1.0 cm quartz cells and a Perkin – Elmer Model
Scheme 1.
561 recorder. All pH measurements were made with a PH-3C digital pH meter (Shanghai Lei Ci Device Works, Shanghai, China) with a combined glass-Calomel electrode.
2.2. Reagents Piperacillin was of analytical reagent grade and purchased from Beijing Chemical Corporation, China. Other chemicals used were of analytical reagent grade. Distilled deionized water was used through out the experiment. The standard stock solution (1 mg/ml) was prepared by dissolving 0.1000 g sodium piperacillin (accurately weighed) with distilled deionized water and dilute it to volume in a 100 ml volumetric flask. The stock solution was protected from light and kept in a refrigerator. The working solution (0.5 mg/ml) were prepared by dilution.
2.3. Procedure Into a 10 ml colorimetric tube were added a certain amount of sodium piperacillin stock solution, 1.5 ml of 2.0 mol/l H2SO4 sequentially. The tube was then diluted to the 5 ml mark with distilled deionized water. The solution was mixed and then degraded in a boiling water-bath for 130 min. After cooling under running water to the room temperature immediately, add 2 mol/l NaOH to adjust the pH value to 7. Another 1.5 ml of 2 mol/l NaOH was added and it was diluted to volume. After shaking, the fluorescent intensity of the solution was measured at 424 nm with excitation at 336 nm against a reagent blank.
2.4. Samples treatment Into a 20 ml centrifuge tube were added 100 mg sodium piperacillin standard solution, 2.5 ml human serum and 5.0 ml 10% trichloroacetic acid in succession, then dilute it with distilled deionized water to the mark. Centrifugalize it for 10 min (3000 rpm). The supernatant was transfered into a separating funnel and extracted three times with
B. Tang et al. / Spectrochimica Acta Part A 57 (2001) 217–222
219
3. Result and discussion
3.1. Excitation and emission spectra
Fig. 1. (a) Excitation and (b) emission spectra of degrading products of piperacillin. Peaks: 1 and 1%, degrading products of piperacillin. 2 and 2%, reagent blank. CPIPC = 0.5 mg/mol; degrading time: 130 min; detection alkalinity: [OH−] =0.3 mol/l; degrading acidity: [H2SO4] = 0.3 mol/l.
In order to determine the optimum working wavelength, the spectral characteristics of degrading products of piperacillin were studied. The corrected excitation and emission spectra (Fig. 1) showed that the wavelengths of maximum excitation and emission of degrading products of PIPC were 336 and 424 nm, respectively.
3.2. Optimization of experimental 6ariables The experimental variables were optimized by applying the univariate method. Degrading time has an important effect on the fluorescence intensity. As can be seen in Fig. 2, the fluorescence intensity varied significantly with changes in degrading time. At the beginning of the reaction, the fluorescence intensity of products increased with the increase of degrading time up to 110 min, then remained constant between 120 and 130 min and decreased thereafter. So, a 130 min degrading time was adopted. The fluorescence intensity of acid degrading products varied with changes in the concentration of H2SO4. Fig. 3 showed that the fluorescence intensity was relatively high and almost remained
Fig. 2. The relationship between relative fluorescence intensity and degrading time. Degrading acidity: [H2SO4] =0.3 mol/l; detection alkalinity: [OH−] = 0.3 mol/l; CPIPC = 0.5 mg/mol.
10 ml portions of ether. Transfer the aqueous layer to a 25 ml volumetric flask and dilute it to the mark.After shaking, keep it in a refrigerator for further use. Divide 10 ml urine of adult men into two portions and add them to 25 ml volumetric flasks, respectively. Into one portion was added 100 mg sodium piperacillin and use the other as a reagent blank. Then dilute them to the mark. After shaking, they were protected from light and kept in a refrigerator.
Fig. 3. The relationship between relative fluorescence intensity and degrading acidity. CPIPC =0.5 mg/mol; detection alkalinity: [OH−]= 0.3 mol/l; degrading time: 130 min.
B. Tang et al. / Spectrochimica Acta Part A 57 (2001) 217–222
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3.3. The stability of the fluorescence intensity of degrading products
Fig. 4. The relationship between relative fluorescence intensity and detection alkalinity. CPIPC = 0.5 mg/mol; degrading time: 130 min; degrading acidity: [H2SO4] =0.3 mol/l.
The stability of the fluorescence intensity of degrading products had an immediate influence on the sensitivity and accuracy. Under the established experimental conditions, make the acid degrading products of 0.5 mg/ml sodium piperacillin exposed to light in light path for various periods of time and then determine the fluorescence intensity. The results are shown in Table 1. From Table 1 it can be seen that within 120 min, the relative deviation of fluorescence measurements was less than 9 2%, which indicated that the degrading products were rather constant. We supposed, with the excitation of strong ultraviolet radiation, initially there was an intramolecular phototautomerism and then partial photolysis happened. Because the ultraviolet intensity indoors was very weak, we ignored the small change of fluorescence intensity during the course of determination. So, fluorometric determination was adaptable to determine degradation products.
3.4. Analytical characteristics constant over the concentration of H2SO4 range 0.2–0.4 mol/l and decreased rapidly outside this concentration range. Therefore, a concentration of H2SO4 of 0.3 mol/l was recommended for use. The influence of detection alkalinity on the fluorescence intensity was also studied under the established experimental conditions. As can be seen in Fig. 4, the fluorescence intensity reached a maximum over the [OH−] range 0.25 – 0.35 mol/l, outside of which smaller intensity was observed. So, 0.3 mol/l was chosen as the detection alkalinity.
Under the optimum experimental conditions, there was a linear relationship between the fluorescence intensity and the concentration of PIPC in the range 7.80–400 ppb with a correlation coefficient (r) of 0.9958. The linear regression equation was DIF = 108.70CPIPC (mg/ml) −1.337. The standard deviation of the fluorescence measurements was 0.085 obtained from a series of 11 blank solutions. The limits of detection (k =3) and of determination (k= 10) of the method were established according to the IUPAC definitions (C1 = KS0/S,where C1 is the limit of detection, S0
Table 1 The quenching of degrading products’ fluorescence Lighting time (min) Fluorescence intensitya RD% a
0 53.00
10 20 30 40 50 60 70 80 90 100 110 52.33 52.63 52.55 52.70 52.93 52.55 52.48 52.63 52.30 52.26 52.30 −1.26 −0.70 −0.84 −0.56 −0.14 −0-84 −0.98 −0.70 −1.26 −1.39 −1.26
Mean value of six measurements.
120 52.40 −1.12
B. Tang et al. / Spectrochimica Acta Part A 57 (2001) 217–222 Table 2 Determination of PIPC in human urine and serum samples (P= 0.95) Samples
Serum Urine a
PIPC added (mg/ml)
0.100 0.100
a
Found (mg/ml) Proposed method
HPLC method [22]
0.09790.004 0.1019 0.001
0.1029 0.003 0.0989 0.005
Each sample was analyzed six times.
the standard error of blank determination, S the slope of the standard curve and K is the constant related to the confidence interval) [35] and the values found were 2.34 and 7.80 ppb, respectively. The relative standard deviation was 2.54%, obtained from a series of ten standards each containing 250 ppb of piperacillin.
3.5. Sample analysis Into a 10 ml colorimetric tube was added 2.5 ml human serum or urine that had been treated.The procedure was used to the determination of trace PIPC in them. The results are given in Table 2.
4. Conclusions
.
From the results presented it can be concluded that the proposed method for the determination of trace PIPC is simple, rapid, stable, sensitive and inexpensive.
Acknowledgements This work was supported by the Natural Science Foundation of China (no. 29975016).
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References [1] L. Verbist, Antimicrob. Ag. Chemother. 13 (1978) 249. [2] A.L. Barry, Cleveland Clin. Quart. 47 (1980) 311. [3] D.A. Leigh, K.T. Simmons, Drugs Exp. Clin. Res. 5 (1979) 99. [4] N.A. Kuck, G.S. Redin, J. Antibiot. 31 (1978) 1175. [5] R.J. Fass, J. Barnishan, Rev. Infect. Dis. 2 (1980) 841. [6] R.J. Fass, Antimicrob. Ag. Chemother. 21 (1982) 1003. [7] G.A. Botta, J.T. Park, J. Bact. 145 (1981) 333. [8] Y.Y. Zhang et al., Antibiotic (China) 7 (6) (1982) 383– 391. [9] Z. Plotkowiak, Z. Seyda, Farm. Pol. 48 (8) (1992) 493– 497. [10] G. Peytavin, A. Baillet, R. Farinotti, J. Pharm. Clin. 10 (1) (1991) 9 – 19. [11] H. Nishi, T. Fukuyama, M. Matsuo, J. Chromatogr. 515 (1990) 245 – 55. [12] M. Park, Y.H. Cho, J. Yang, Yakhak Hoechi, 28 (1) (1984) 25 – 7. [13] R. Wang, X. Sun, Chin. J. Pharm. Anal. 11 (5) (1991) 282 – 284. [14] S. Kumiko, S. Toru et al., Chemotherapy (Tokyo) 42 (Suppl. 2) (1994) 263 – 76. [15] W. Cullmann, W. Dick, M. Edelmann, J. Clin. Chem. Clin. Biochem. 23(3) (1985) 151 – 6. [16] B. Pospisilova, M. Simkova, Kubes, J. Pharmazie. 41 (10) (1986) 705 – 8. [17] B. Pospisilova, M. Simkova, Kubes, J. Cesk. Farm. 34 (3 – 4) 1085. [18] N. Abo El-Maali, M.A. Ghandour, M. Khodari, Talanta, 40 (12) (1993) 1833 – 1838. [19] L. Campanella, F. Mazzei et al., Ann. Chim. (Rome) 80 (9 – 10) (1990) 395 – 412. [20] L. Campanella, F. Mazzei et al., J. Pharm. Biomed. Anal. 6 (3) (1988) 299 – 305. [21] S. Schroeder, R. Voigt et al., Pharmazie. 40 (5) (1985) 333 – 5. [22] J.C. Garcia-Glez, R. Mendez et al., J. Chromatogr. A 812 (1 – 2) (1998) 213 – 220. [23] M.A. Campanero, A.M. Zamarreno et al. Chromatographia 46 (7 – 8) (1997) 374 – 380. [24] T. Tsukamoto, T. Ushio, J. Chromatogr. A 678 (1) (1994) 69 – 76. [25] S.L. Qiu, J.H. Liu, Chin. J. Pharm. Anal. 9 (1) (1989) 27 – 9, 17. [26] V. Gautier, F. Demotes-Mainard, J. Pharm. Biomed. Anal. 9 (2) (1991) 183 – 186. [27] A.P. Ocampo, K.O. Hoyt et al., J. Chromatogr. 496 (1989) 167. [28] C.E. Parker, J.R. Perkins et al., J. Chromatogr., Biomed. Appl. 127 (1993) 45 – 57. [29] A. Zarzuelo, F.G. Lopez et al., J. Liq. Chromatogr. Relat. Technol. 19 (4) (1996) 601 – 610. [30] K. Iwaki, N. Okumura et al. J. Chromatogr. 504(2) (1990) 359 – 67.
222
B. Tang et al. / Spectrochimica Acta Part A 57 (2001) 217–222
[31] T. Marunaka, M. Maniwa et al., J. Chromatogr., Biomed. Appl. 75 (1988) 87–101. [32] D.K. An, Pharmaceutical Analysis, second ed, Beijing Press of People Hygiene, 1991, p. 334. [33] B. Tang, X.W. He, H.X. Shen, Chin. J. Anal. Chem. 22 (11) (1994) 1089.
.
[34] X.W. He, B. Tang, H.X. Shen, Chem. J. Chin. Uni. 16 (1) (1995) 26. [35] H.M.N.H. Irving, H. Freiser, T.S. West, (Eds.) IUPAC Compendium of Analytical Nomenclature, Definitive Rules, Pergamon Press, Oxford, 1981.