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Spectrofluorimetric determination of cisatracurium and mivacurium in spiked human serum and pharmaceuticals
Talanta 49 (1999) 881 – 887
Spectrofluorimetric determination of cisatracurium and mivacurium in spiked human serum and pharmaceuticals Rut Ferna´ndez, Miguel Angel Bello *, Manuel Callejo´n, Juan Carlos Jime´nez, Alfonso Guirau´m Department of Analytical Chemistry, Faculty of Chemistry, 41012 -Se6ille, Spain Received 12 October 1998; received in revised form 9 February 1999; accepted 26 February 1999
Abstract Spectrofluorimetric methods to determine cisatracurium and mivacurium are proposed and applied to the determination of both substances in human serum and to the determination of mivacurium in pharmaceuticals. The fluorimetric methods allow the determination of 5 – 500 ng ml − 1 of mivacurium in aqueous solutions and 5 – 500 ng ml − 1 of cisatracurium in water–acetonitrile solutions, both containing acetic acid – sodium acetate buffer (pH 5.5) with lexc =230 nm and lem =324 nm. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Cisatracurium determination; Fluorescence; Mivacurium determination; Serum
1. Introduction Cisatracurium besylate {(1R,1%R,2R,2%R)-2,2[1,5-pentanediylbis-[oxy(3-oxo-3,1-propanediyl)]]bis[1 - [(3,4 - dimethoxyphenyl) - methyl] - 1,2,3,4 tetrahydro - 6,7 - dimethoxy - 2 - methylisoquinolinium] dibenzene sulfonate} (Fig. 1) and mivacurium {(E)-(1R,1%R)-,2%-[4-octenedioyl-bis(oxytrimethyllene)]bis[1,2,3,4-tetrahydro-6,7-dimethoxy2 - methyl - 1 - (3,4,5 - trimethoxybenzyl) - isoquinolium] dichloride} (Fig. 2) are non-depolarizing
* Corresponding author. Tel.: +34-954557172; fax: + 34954557168. E-mail address: [email protected] (M.A. Bello)
neuromuscular blocking agents. Mivacurium consists of a mixture of three stereoisomers. Cisatracurium is one of ten stereoisomers (R-cis, R%-cis) that comprise atracurium which represents approximately 15% of the atracurium mixture and is approximately 3-fold more potent than the mixture that constitutes the parent drug [1]. Several reports describe high-performance liquid chromatography (HPLC) methods for the analysis of plasma concentrations of cisatracurium alone using either ultraviolet [2] or mass spectrometry detection [3]; ion chromatography with fluorescence detection [4] also has been used. For mivacurium, HPLC methods with ultraviolet [5] and fluorescence detection [6–9] have been described.
0039-9140/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 9 - 9 1 4 0 ( 9 9 ) 0 0 0 8 4 - 3
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R. Ferna´ndez et al. / Talanta 49 (1999) 881–887
This work constitutes the first stage of a research schedule focused on the proposal of new analytical procedures for the determination of cisatracurium and mivacurium as alternatives to the HPLC methods. This paper describes the spectrofluorimetric determination of cisatracurium and mivacurium in serum and also has been applied to the determination in pharmaceuticals.
2. Experimental
2.1. Reagents Cisatracurium besylate and mivacurim were kindly provided by Glaxo-Wellcome (Greenford, Middlesex, UK). Acetic acid, sodium acetate, picric acid, acetonitrile, sulfuric acid and isopropanol were of analytical-reagent grade and purchased from Merck (Darmstadt, Germany); dichloromethane of HPLC grade was obtained from Romil (Cambridge, UK). High-purity water was obtained from a Millipore (Milford, MA, USA) Milli-Q Plus system. Stock standard mivacurium and cisatracurium solutions of 100 mg ml − 1 and working standard solution of 1 mg ml − 1 were prepared by dissolving the corresponding substance in high-purity water; both solutions were stable for several months at room temperature. Serum pooled samples were obtained from healthy volunteers. To adjust the pH of the solutions, an acetic acid – sodium acetate 0.1 M pH 5.5 buffer was used. Picric acid solution was prepared by dilution 1:50 (v/v) from aqueous saturated picric acid solution.
2.2. Apparatus Fluorescence intensity was measured on a Perkin-Elmer (Norwalk, CT, USA) LS-5 luminescence spectrometer equipped with a xenon lamp and an Acer Model 1030 computer working with the FLUORPACK software from Sciware (Mallorca, Spain). All measurements were performed in a standard 10-mm pathlength quartz cell, thermostatted at 25.09 0.5°C, with 5 nm bandwidths for the emission and excitation monochromators. A Philips (Eindoven, Netherlands) Model PU8720 UV/VIS spectrophotometer was used for the absorbance measurements. The pH was measured on a Crison (Barcelona, Spain) MicropH 2002 pH-meter. Centrifugation of serum samples was carried out with a Sigma (Osterode, Germany) Laborzentrifugen 4-10. For agitation in the serum extraction procedure, a Selecta (Barcelona, Spain) Vibromatic 384 shaker was used.
2.3. Spectrofluorimetric determination of cisatracurium Into 25-ml calibrated flasks were pipetted suitable aliquots of working solution of cisatracurium containing 125–12 500 ng of cisatracurium; 5 ml of 0.1 M acetic acid–sodium acetate buffer (pH 5.5) and 5 ml of acetonitrile were added and diluted to the mark with water. The solutions were thermostatted at 259 0.1°C and the fluorescence measured at 324 nm using an excitation wavelength of 230 nm against a blank solution. The concentration of cisatracurium in the sample was determined from a calibration graph prepared
Fig. 1. Chemical formula of cisatracurium besylate.
R. Ferna´ndez et al. / Talanta 49 (1999) 881–887
883
Fig. 2. Chemical formula of mivacurium dichloride.
under identical conditions. The prepared solutions remain stable for at least 24 h.
2.4. Spectrofluorimetric determination of mi6acurium Into 25-ml calibrated flasks were pipetted suitable aliquots of working solution of mivacurium containing 125 – 12 500 ng of mivacurium; 5 ml of 0.1 M acetic acid – sodium acetate buffer (pH 5.5) was added and diluted to the mark with water. Solutions were thermostatted at 25 90.1°C and the fluorescence was measured at 324 nm using an excitation wavelength of 230 nm against a blank solution. The concentration of mivacurium in the sample was determined from a calibration graph prepared under identical conditions. The prepared solutions remain stable for at least 24 h.
2.5. Samples The proposed procedure for the determination of mivacurium was applied to the direct determination in one Spanish commercialized pharmaceutical formulation (Mivacron® injection blisters). The method was also applied to the determination of mivacurium and cisatracurium in spiked human pooled sera which were kindly provided from hospitals in the city.
2.5.1. Serum preparation Serum (0.5 ml) spiked with a suitable quantity of cisatracurium or mivacurium was poured into a 15-ml centrifuge tube with a thread lock and then 0.25 ml of picric acid solution, three drops of
sulfuric acid 1:2 (v/v), 0.4 ml of isopropanol and 2.1 ml of dichloromethane were added. The tube was vigorously shaken in a mechanical shaker for 10 min and then centrifuged (6000 × g) for 5 min. The organic layer was transferred to a reservoir for collecting subsequent organic phases. The aqueous phase was treated with 1 ml of dichloromethane, agitated for 10 min by the mechanical shaker and centrifuged for 5 min. The last organic extract was also transferred to the reservoir and all the combined organic extracts were then evaporated to dryness under a nitrogen stream. The tube was removed immediately after drying and the solution reconstituted with 5 ml of acetic acid–sodium acetate buffer. The sample was then treated and measured according to the spectrofluorimetric determination procedures described in Sections 2.3 and 2.4. A blank was prepared under the same conditions but without cisatracurium or mivacurium spiking. Samples prepared from sera must be filtered (0.45 mm) prior to fluorescence intensity measurements.
3. Results and discussion
3.1. Study of the emission features of the cisatracurium and mi6acurium solutions Fig. 3(A) shows the excitation spectra for aqueous solutions of cisatracurium and mivacurium, in which a maximum at 230 nm can be clearly observed for both substances. Fig. 3(B) shows the emission spectra for aqueous cisatracurium and mivacurium using lexc = 230 nm; a
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R. Ferna´ndez et al. / Talanta 49 (1999) 881–887
Fig. 3. Excitation spectra (A) and emission spectra with lexc = 230 nm (B) for aqueous solutions of cisatracurium (C) and mivacurium (M).
maximum at 324 nm can be observed for both substances. This is in agreement with the similar molecular structures of cisatracurium and mivacurium. The influence of pH on the fluorescence intensity of cisatracurium and mivacurium solutions can be observed in Fig. 4. Using as excitation wavelength 230 nm, the fluorescence intensity measured at 324 nm exhibit a maximum within a
pH range of 3.5–6 for cisatracurium and 4.4–10.7 for mivacurium. Thus, an acetic acid–sodium acetate buffer solution of pH 5.5 was chosen for the spectrofluorimetric determination of cisatracurium and mivacurium. The effect of the concentration of the buffer solution was studied. Fluorescence intensity remains stable for total buffer concentrations lower than 4×10 − 2 M and 2×10 − 2 M for cisatracurium and mivacurium, respectively; higher buffer concentrations lead to fluorescence decay. Accordingly, a 5-ml aliquot of 0.1 M buffer solution for 25 ml of total volume was selected as a suitable volume for the recommended procedure. With the aim to study the influence of the ionic strength, aqueous solutions of cisatracurium and mivacurium containing buffer at various concentrations of KCl were prepared. The results obtained show that no significant changes occur in the fluorescence intensity of cisatracurium and mivacurium for concentrations between 0 and 1 M KCl. The effect of the presence of organic miscible solvents was tested for methanol, ethanol and acetonitrile. For cisatracurium, an important increase in the fluorescence intensity was noticeable
Fig. 4. Influence of pH on the fluorescence intensity for cisatracurium (A) and mivacurium (B) solutions (lexc =230 nm; lem =324 nm).
R. Ferna´ndez et al. / Talanta 49 (1999) 881–887
885
Fig. 5. Emission spectra (lexc = 230 nm) for blank serum (B) and for sera spiked with cisatracurium (C) and mivacurium (M).
when water-miscible organic solvents were added. The best results were found for acetonitrile, yielding an increase of 100% in the fluorescence intensity for an addition of 20% (v/v) of this solvent. Poor increases in the fluorescence intensity were found for mivacurium even at higher organic solvent concentrations. The influence of the temperature on the fluorescence intensity shows a nearlly linear (negative) relationship between temperature and fluorescence intensity for cisatracurium and mivacurium. When the temperature is decreased the fluorescence is enhanced, as expected. Hence samples were thermostatted at 259 0.5°C in the proposed procedure.
3.2. Selecti6ity: effect of interfering substances A study of some potential interferences in the spectrofluorimetric determination of mivacurium and cisatracurium was performed by selecting the excipients often used in pharmaceutical formulations or as possible co-active substance. Samples containing a fixed amount of cisatracurium or mivacurium (100 ng ml − 1) and variable concentrations of potential interfering compounds were measured. Lactose, sucrose,
glucose, fructose and succinylcholine did not cause interference at weight ratios (interfering substance/active substance) 5 10 000, and 5 5000 for succinylcholine/mivacurium. Metamizole causes interference for ratios ] 15 for cisatracurium and ]1 for mivacurium. Ratios 5 20 for bupivacaine/mivacurium and 5 1 for bupivacaine/cisatracurium did not cause interference.
3.3. Linearity of the response A series of standard solutions (five replicates) of cisatracurium and mivacurium were prepared by following the procedures described in Section 2. The calibration graphs of fluorescence intensity (y) versus cisatracurium or mivacurium concentration (x) were found to be linear over the range 5.0–500.0 ng ml − 1 for cisatracurium (Eq. (1)) and mivacurium (Eq. (2)): y=(− 0.19 0.3)+ (0.4299 0.002)x (n= 10, r= 0.9997)
(1)
y=(0.09 0.2)+ (0.4269 0.001)x (n= 10, r= 0.9998)
(2)
R. Ferna´ndez et al. / Talanta 49 (1999) 881–887
886
The application of Student’s t-test shows that the intercepts are non-significant and accordingly the straight lines pass through the origin.
This leads to a quantitation limit of 2.1 ng ml − 1 for cisatracurium and 1.4 ng ml − 1 for mivacurium.
3.4. Precision
3.6. Validation of the spectrofluorimetric determinations of cisatracurium and mi6acurium
Eleven replicates carried out on different days within a month of 100 ng ml − 1 target solutions of cisatracurium and mivacurium were made by using the proposed procedures. The results were 99.890.3 and 100.1 90.2, respectively, which leads to a R.S.D. between-day precision of 0.3% and 0.2%, respectively.
3.5. Detection and quantitation limit According to the Analytical Methods Committee [11], the detection limit (LOD) is the concentration of cisatracurium or mivacurium corresponding to a signal equal to the blank mean (yB) plus three times the standard deviation of the blank (sB). Eleven blank measurements gave an average signal blank yB =0.2 and a standard deviation sB =0.1 for the cisatracurium blank and yB =0.1 and sB =0.1 for the mivacurium blank. Thus, the analytical signal corresponding to LOD is yB +3sB. This value is transformed in LOD through the equations of the calibration lines, giving 1.39 ng ml − 1 for cisatracurium and 0.93 ng ml − 1 for mivacurium. From the calibration straight lines it is also possible to estimate the quantitation limits as the concentration corresponding to the ratio between three times the S.D. of the intercept and the slope of the calibration line [12].
3.6.1. Analysis of pharmaceutical samples Five injection blisters of Mivacrom® (GlaxoWellcome) with a mivacurium label content of 2 mg ml − 1 were directly analyzed with the proposed method from external calibration obtaining a result of 1.99 0.2 mg ml − 1 of mivacurium. The application of an independent method (direct spectrophotometric determination at 280.7 nm) shows a result of 2.09 0.2 mg ml − 1; as can be observed, there is good agreement between both methods. 3.6.2. Analysis of spiked sera Serum samples were spiked with cisatracurium or mivacurium to obtain concentrations in serum of 5 and 10 mg ml − 1 and treated according to the above described preparation and spectrofluorimetric determination procedures; the final solutions measured contained 100 and 200 ng ml − 1 of the active substance, respectively. Fig. 5 shows the emission spectra with lexc = 230 nm for the samples obtained from blank serum (B) and from sera spiked with cisatracurium (C) and mivacurium (M). Poor recoveries were found for mivacurium, ranging from 65 to 70%, values very similar to those obtained by other authors [6]. In order to avoid matrix effects that affect the results, the standard additions method [10] was used for mi-
Table 1 Results for the analyzed samples of spiked sera Substance
Spiked concentration (mg ml−1)
HPLC method (mg ml−1)a,b
Proposed method (mg ml−1)b
Cisatracurium
5.0 10.0 5.0 10.0
4.9 9 0.2 10.2 90.1 5.0 90.1 10.0 90.2
4.8 90.3 10.2 90.3 4.9 90.2 10.2 90.1
Mivacurium
a b
For details of the method see text. Average of five determinations9 S.D.
R. Ferna´ndez et al. / Talanta 49 (1999) 881–887
vacurium. For cisatracurium it is possible to apply external calibration. The samples were also analyzed by HPLC methods using UV detection at 280 nm for cisatracurium [2] and at 210 nm for mivacurium [5]. The results obtained are collected in Table 1. As can be seen, good agreement was found between the HPLC and the proposed methods (statistically proved according to the paired ttest [12]) with respect to the spiked concentration (the application of Student’s t-test indicates that the method is accurate (null hypothesis accepted) [12]).
4. Conclusions The results obtained show that the proposed method may be useful to determine cisatracurium or mivacurium in spiked human serum at the levels obtained after the administration of normal clinical doses, and it would be a method of choice for monitoring these substances in patients. The method for mivacurium has also been applied to the determination of the active constituent in a commercial pharmaceutical preparation.
887
Acknowledgements The authors gratefully acknowledge Glaxo-Wellcome for supplying cisatracurium and mivacurium.
References [1] A. Bryson, D. Faulds, Drugs 53 (5) (1997) 848. [2] H. Zhang, P. Wang, M.G. Bartlett, J.T. Stewart, J. Pharm. Biomed. Anal. 16 (7) (1998) 1241. [3] G.J. Dear, J.C. Harrelson, A.E. Jones, T.E. Johnson, S. Pleasance, Rapid Commun. Mass Spectrom. 9 (14) (1995) 1457. [4] B.J. Bryant, C.D. James, D.R. Cook, J.C. Harrelson, J. Liq. Chromatogr. 20 (13) (1997) 2041. [5] R. DeAngelis, P. Loebs, R. Maehr, J. Savarese, R.M. Welch, J. Chromatogr. 525 (1990) 389. [6] A. Brown, C. James, R. Welch, J. Harrelson, J. Chromatogr. Biomed. Appl. 578 (1992) 302. [7] W. Biederbick, G. Aydinciouglou, C. Diefenbach, M. Theisohn, J. Chromatogr. Biomed. Appl. 685 (1996) 315. [8] M. Weindlmayr-Goettel, G. Weberhofer, H. Gilly, H.G. Kress, J. Chromatogr. Biomed. Appl. 685 (1996) 123. [9] S.I. Lugo, N.D. Eddington, J. Pharm. Biomed. Anal. 14 (6) (1996) 675. [10] L. Cuadros, A.M. Garcı´a, M. Garcı´a Campan˜a, F. Ale´s, C. Jime´nez, M. Roma´n Ceba, J. AOAC Int. 78 (1995) 471. [11] Analytical Methods Committee, Analyst 112 (1987) 199. [12] J.C. Miller, J.N. Miller, Statistics for Analytical Chemistry, 3rd ed., Ellis Horwood – Prentice Hall, Chichester, 1993.
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Spectrofluorimetric determination of cisatracurium and mivacurium in spiked human serum and pharmaceuticals Rut Ferna´ndez, Miguel Angel Bello *, Manuel Callejo´n, Juan Carlos Jime´nez, Alfonso Guirau´m Department of Analytical Chemistry, Faculty of Chemistry, 41012 -Se6ille, Spain Received 12 October 1998; received in revised form 9 February 1999; accepted 26 February 1999
Abstract Spectrofluorimetric methods to determine cisatracurium and mivacurium are proposed and applied to the determination of both substances in human serum and to the determination of mivacurium in pharmaceuticals. The fluorimetric methods allow the determination of 5 – 500 ng ml − 1 of mivacurium in aqueous solutions and 5 – 500 ng ml − 1 of cisatracurium in water–acetonitrile solutions, both containing acetic acid – sodium acetate buffer (pH 5.5) with lexc =230 nm and lem =324 nm. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Cisatracurium determination; Fluorescence; Mivacurium determination; Serum
1. Introduction Cisatracurium besylate {(1R,1%R,2R,2%R)-2,2[1,5-pentanediylbis-[oxy(3-oxo-3,1-propanediyl)]]bis[1 - [(3,4 - dimethoxyphenyl) - methyl] - 1,2,3,4 tetrahydro - 6,7 - dimethoxy - 2 - methylisoquinolinium] dibenzene sulfonate} (Fig. 1) and mivacurium {(E)-(1R,1%R)-,2%-[4-octenedioyl-bis(oxytrimethyllene)]bis[1,2,3,4-tetrahydro-6,7-dimethoxy2 - methyl - 1 - (3,4,5 - trimethoxybenzyl) - isoquinolium] dichloride} (Fig. 2) are non-depolarizing
* Corresponding author. Tel.: +34-954557172; fax: + 34954557168. E-mail address: [email protected] (M.A. Bello)
neuromuscular blocking agents. Mivacurium consists of a mixture of three stereoisomers. Cisatracurium is one of ten stereoisomers (R-cis, R%-cis) that comprise atracurium which represents approximately 15% of the atracurium mixture and is approximately 3-fold more potent than the mixture that constitutes the parent drug [1]. Several reports describe high-performance liquid chromatography (HPLC) methods for the analysis of plasma concentrations of cisatracurium alone using either ultraviolet [2] or mass spectrometry detection [3]; ion chromatography with fluorescence detection [4] also has been used. For mivacurium, HPLC methods with ultraviolet [5] and fluorescence detection [6–9] have been described.
0039-9140/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 9 - 9 1 4 0 ( 9 9 ) 0 0 0 8 4 - 3
882
R. Ferna´ndez et al. / Talanta 49 (1999) 881–887
This work constitutes the first stage of a research schedule focused on the proposal of new analytical procedures for the determination of cisatracurium and mivacurium as alternatives to the HPLC methods. This paper describes the spectrofluorimetric determination of cisatracurium and mivacurium in serum and also has been applied to the determination in pharmaceuticals.
2. Experimental
2.1. Reagents Cisatracurium besylate and mivacurim were kindly provided by Glaxo-Wellcome (Greenford, Middlesex, UK). Acetic acid, sodium acetate, picric acid, acetonitrile, sulfuric acid and isopropanol were of analytical-reagent grade and purchased from Merck (Darmstadt, Germany); dichloromethane of HPLC grade was obtained from Romil (Cambridge, UK). High-purity water was obtained from a Millipore (Milford, MA, USA) Milli-Q Plus system. Stock standard mivacurium and cisatracurium solutions of 100 mg ml − 1 and working standard solution of 1 mg ml − 1 were prepared by dissolving the corresponding substance in high-purity water; both solutions were stable for several months at room temperature. Serum pooled samples were obtained from healthy volunteers. To adjust the pH of the solutions, an acetic acid – sodium acetate 0.1 M pH 5.5 buffer was used. Picric acid solution was prepared by dilution 1:50 (v/v) from aqueous saturated picric acid solution.
2.2. Apparatus Fluorescence intensity was measured on a Perkin-Elmer (Norwalk, CT, USA) LS-5 luminescence spectrometer equipped with a xenon lamp and an Acer Model 1030 computer working with the FLUORPACK software from Sciware (Mallorca, Spain). All measurements were performed in a standard 10-mm pathlength quartz cell, thermostatted at 25.09 0.5°C, with 5 nm bandwidths for the emission and excitation monochromators. A Philips (Eindoven, Netherlands) Model PU8720 UV/VIS spectrophotometer was used for the absorbance measurements. The pH was measured on a Crison (Barcelona, Spain) MicropH 2002 pH-meter. Centrifugation of serum samples was carried out with a Sigma (Osterode, Germany) Laborzentrifugen 4-10. For agitation in the serum extraction procedure, a Selecta (Barcelona, Spain) Vibromatic 384 shaker was used.
2.3. Spectrofluorimetric determination of cisatracurium Into 25-ml calibrated flasks were pipetted suitable aliquots of working solution of cisatracurium containing 125–12 500 ng of cisatracurium; 5 ml of 0.1 M acetic acid–sodium acetate buffer (pH 5.5) and 5 ml of acetonitrile were added and diluted to the mark with water. The solutions were thermostatted at 259 0.1°C and the fluorescence measured at 324 nm using an excitation wavelength of 230 nm against a blank solution. The concentration of cisatracurium in the sample was determined from a calibration graph prepared
Fig. 1. Chemical formula of cisatracurium besylate.
R. Ferna´ndez et al. / Talanta 49 (1999) 881–887
883
Fig. 2. Chemical formula of mivacurium dichloride.
under identical conditions. The prepared solutions remain stable for at least 24 h.
2.4. Spectrofluorimetric determination of mi6acurium Into 25-ml calibrated flasks were pipetted suitable aliquots of working solution of mivacurium containing 125 – 12 500 ng of mivacurium; 5 ml of 0.1 M acetic acid – sodium acetate buffer (pH 5.5) was added and diluted to the mark with water. Solutions were thermostatted at 25 90.1°C and the fluorescence was measured at 324 nm using an excitation wavelength of 230 nm against a blank solution. The concentration of mivacurium in the sample was determined from a calibration graph prepared under identical conditions. The prepared solutions remain stable for at least 24 h.
2.5. Samples The proposed procedure for the determination of mivacurium was applied to the direct determination in one Spanish commercialized pharmaceutical formulation (Mivacron® injection blisters). The method was also applied to the determination of mivacurium and cisatracurium in spiked human pooled sera which were kindly provided from hospitals in the city.
2.5.1. Serum preparation Serum (0.5 ml) spiked with a suitable quantity of cisatracurium or mivacurium was poured into a 15-ml centrifuge tube with a thread lock and then 0.25 ml of picric acid solution, three drops of
sulfuric acid 1:2 (v/v), 0.4 ml of isopropanol and 2.1 ml of dichloromethane were added. The tube was vigorously shaken in a mechanical shaker for 10 min and then centrifuged (6000 × g) for 5 min. The organic layer was transferred to a reservoir for collecting subsequent organic phases. The aqueous phase was treated with 1 ml of dichloromethane, agitated for 10 min by the mechanical shaker and centrifuged for 5 min. The last organic extract was also transferred to the reservoir and all the combined organic extracts were then evaporated to dryness under a nitrogen stream. The tube was removed immediately after drying and the solution reconstituted with 5 ml of acetic acid–sodium acetate buffer. The sample was then treated and measured according to the spectrofluorimetric determination procedures described in Sections 2.3 and 2.4. A blank was prepared under the same conditions but without cisatracurium or mivacurium spiking. Samples prepared from sera must be filtered (0.45 mm) prior to fluorescence intensity measurements.
3. Results and discussion
3.1. Study of the emission features of the cisatracurium and mi6acurium solutions Fig. 3(A) shows the excitation spectra for aqueous solutions of cisatracurium and mivacurium, in which a maximum at 230 nm can be clearly observed for both substances. Fig. 3(B) shows the emission spectra for aqueous cisatracurium and mivacurium using lexc = 230 nm; a
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R. Ferna´ndez et al. / Talanta 49 (1999) 881–887
Fig. 3. Excitation spectra (A) and emission spectra with lexc = 230 nm (B) for aqueous solutions of cisatracurium (C) and mivacurium (M).
maximum at 324 nm can be observed for both substances. This is in agreement with the similar molecular structures of cisatracurium and mivacurium. The influence of pH on the fluorescence intensity of cisatracurium and mivacurium solutions can be observed in Fig. 4. Using as excitation wavelength 230 nm, the fluorescence intensity measured at 324 nm exhibit a maximum within a
pH range of 3.5–6 for cisatracurium and 4.4–10.7 for mivacurium. Thus, an acetic acid–sodium acetate buffer solution of pH 5.5 was chosen for the spectrofluorimetric determination of cisatracurium and mivacurium. The effect of the concentration of the buffer solution was studied. Fluorescence intensity remains stable for total buffer concentrations lower than 4×10 − 2 M and 2×10 − 2 M for cisatracurium and mivacurium, respectively; higher buffer concentrations lead to fluorescence decay. Accordingly, a 5-ml aliquot of 0.1 M buffer solution for 25 ml of total volume was selected as a suitable volume for the recommended procedure. With the aim to study the influence of the ionic strength, aqueous solutions of cisatracurium and mivacurium containing buffer at various concentrations of KCl were prepared. The results obtained show that no significant changes occur in the fluorescence intensity of cisatracurium and mivacurium for concentrations between 0 and 1 M KCl. The effect of the presence of organic miscible solvents was tested for methanol, ethanol and acetonitrile. For cisatracurium, an important increase in the fluorescence intensity was noticeable
Fig. 4. Influence of pH on the fluorescence intensity for cisatracurium (A) and mivacurium (B) solutions (lexc =230 nm; lem =324 nm).
R. Ferna´ndez et al. / Talanta 49 (1999) 881–887
885
Fig. 5. Emission spectra (lexc = 230 nm) for blank serum (B) and for sera spiked with cisatracurium (C) and mivacurium (M).
when water-miscible organic solvents were added. The best results were found for acetonitrile, yielding an increase of 100% in the fluorescence intensity for an addition of 20% (v/v) of this solvent. Poor increases in the fluorescence intensity were found for mivacurium even at higher organic solvent concentrations. The influence of the temperature on the fluorescence intensity shows a nearlly linear (negative) relationship between temperature and fluorescence intensity for cisatracurium and mivacurium. When the temperature is decreased the fluorescence is enhanced, as expected. Hence samples were thermostatted at 259 0.5°C in the proposed procedure.
3.2. Selecti6ity: effect of interfering substances A study of some potential interferences in the spectrofluorimetric determination of mivacurium and cisatracurium was performed by selecting the excipients often used in pharmaceutical formulations or as possible co-active substance. Samples containing a fixed amount of cisatracurium or mivacurium (100 ng ml − 1) and variable concentrations of potential interfering compounds were measured. Lactose, sucrose,
glucose, fructose and succinylcholine did not cause interference at weight ratios (interfering substance/active substance) 5 10 000, and 5 5000 for succinylcholine/mivacurium. Metamizole causes interference for ratios ] 15 for cisatracurium and ]1 for mivacurium. Ratios 5 20 for bupivacaine/mivacurium and 5 1 for bupivacaine/cisatracurium did not cause interference.
3.3. Linearity of the response A series of standard solutions (five replicates) of cisatracurium and mivacurium were prepared by following the procedures described in Section 2. The calibration graphs of fluorescence intensity (y) versus cisatracurium or mivacurium concentration (x) were found to be linear over the range 5.0–500.0 ng ml − 1 for cisatracurium (Eq. (1)) and mivacurium (Eq. (2)): y=(− 0.19 0.3)+ (0.4299 0.002)x (n= 10, r= 0.9997)
(1)
y=(0.09 0.2)+ (0.4269 0.001)x (n= 10, r= 0.9998)
(2)
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886
The application of Student’s t-test shows that the intercepts are non-significant and accordingly the straight lines pass through the origin.
This leads to a quantitation limit of 2.1 ng ml − 1 for cisatracurium and 1.4 ng ml − 1 for mivacurium.
3.4. Precision
3.6. Validation of the spectrofluorimetric determinations of cisatracurium and mi6acurium
Eleven replicates carried out on different days within a month of 100 ng ml − 1 target solutions of cisatracurium and mivacurium were made by using the proposed procedures. The results were 99.890.3 and 100.1 90.2, respectively, which leads to a R.S.D. between-day precision of 0.3% and 0.2%, respectively.
3.5. Detection and quantitation limit According to the Analytical Methods Committee [11], the detection limit (LOD) is the concentration of cisatracurium or mivacurium corresponding to a signal equal to the blank mean (yB) plus three times the standard deviation of the blank (sB). Eleven blank measurements gave an average signal blank yB =0.2 and a standard deviation sB =0.1 for the cisatracurium blank and yB =0.1 and sB =0.1 for the mivacurium blank. Thus, the analytical signal corresponding to LOD is yB +3sB. This value is transformed in LOD through the equations of the calibration lines, giving 1.39 ng ml − 1 for cisatracurium and 0.93 ng ml − 1 for mivacurium. From the calibration straight lines it is also possible to estimate the quantitation limits as the concentration corresponding to the ratio between three times the S.D. of the intercept and the slope of the calibration line [12].
3.6.1. Analysis of pharmaceutical samples Five injection blisters of Mivacrom® (GlaxoWellcome) with a mivacurium label content of 2 mg ml − 1 were directly analyzed with the proposed method from external calibration obtaining a result of 1.99 0.2 mg ml − 1 of mivacurium. The application of an independent method (direct spectrophotometric determination at 280.7 nm) shows a result of 2.09 0.2 mg ml − 1; as can be observed, there is good agreement between both methods. 3.6.2. Analysis of spiked sera Serum samples were spiked with cisatracurium or mivacurium to obtain concentrations in serum of 5 and 10 mg ml − 1 and treated according to the above described preparation and spectrofluorimetric determination procedures; the final solutions measured contained 100 and 200 ng ml − 1 of the active substance, respectively. Fig. 5 shows the emission spectra with lexc = 230 nm for the samples obtained from blank serum (B) and from sera spiked with cisatracurium (C) and mivacurium (M). Poor recoveries were found for mivacurium, ranging from 65 to 70%, values very similar to those obtained by other authors [6]. In order to avoid matrix effects that affect the results, the standard additions method [10] was used for mi-
Table 1 Results for the analyzed samples of spiked sera Substance
Spiked concentration (mg ml−1)
HPLC method (mg ml−1)a,b
Proposed method (mg ml−1)b
Cisatracurium
5.0 10.0 5.0 10.0
4.9 9 0.2 10.2 90.1 5.0 90.1 10.0 90.2
4.8 90.3 10.2 90.3 4.9 90.2 10.2 90.1
Mivacurium
a b
For details of the method see text. Average of five determinations9 S.D.
R. Ferna´ndez et al. / Talanta 49 (1999) 881–887
vacurium. For cisatracurium it is possible to apply external calibration. The samples were also analyzed by HPLC methods using UV detection at 280 nm for cisatracurium [2] and at 210 nm for mivacurium [5]. The results obtained are collected in Table 1. As can be seen, good agreement was found between the HPLC and the proposed methods (statistically proved according to the paired ttest [12]) with respect to the spiked concentration (the application of Student’s t-test indicates that the method is accurate (null hypothesis accepted) [12]).
4. Conclusions The results obtained show that the proposed method may be useful to determine cisatracurium or mivacurium in spiked human serum at the levels obtained after the administration of normal clinical doses, and it would be a method of choice for monitoring these substances in patients. The method for mivacurium has also been applied to the determination of the active constituent in a commercial pharmaceutical preparation.
887
Acknowledgements The authors gratefully acknowledge Glaxo-Wellcome for supplying cisatracurium and mivacurium.
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