Anal. Chem. 1997, 69, 1364-1369
Evaluation of Capillary Zone Electrophoresis and Micellar Electrokinetic Capillary Chromatography with Direct Injection of Plasma for the Determination of Cefotaxime and Its Metabolite G. Castaneda Penalvo,† M. Kelly,‡ H. Maillols,§ and H. Fabre*,⊥
Laboratoire de Chimie Analytique, and Laboratoire de Technique Pharmaceutique Industrielle, Faculte´ de Pharmacie, Universite´ Montpellier I, 34060 Montpellier, France, Departamento de Quimica Analitica y Tecnologia de los Alimentos, Universidad de Castilla La Mancha, Ciudad Real, Spain, and Department of Chemistry, Royal College of Surgeons in Ireland, Dublin 2, Ireland
Quantitative aspects of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) were investigated for the determination of cefotaxime (C) and its deacetyl metabolite (DA) in human plasma in a concentration range of therapeutic interest. For CZE, plasma samples spiked with C and DA were injected after deproteinization with acetonitrile, and analytes were separated in a fused silica capillary using a borate buffer at pH 9.2 as electrolyte; no suitable internal standard was found. For MECC, plasma samples spiked with C, DA, and theobromine as internal standard were directly injected after dilution with water and analyzed using a phosphate buffer, pH 8.00, containing 165 mM SDS as separation electrolyte and a fused silica capillary. Both methods gave satisfactory interday precision with respect to migration times (RSD 0.995), with an intraday precision at each concentration better than 10% (Table 1). However, linearity experiments carried out on each of the four days showed a great variability for C, with a RSD of 16.1% between the slopes. It is thought that this variability originated from fluctuations in the day-to-day temperature within the laboratory which influenced the evaporation of the acetonitrile and which is not compensated for by an internal standard. Limits of Detection (LOD) and Quantification (LOQ). The LOD for C and DA based on a signal-to-noise ratio of 3 was about 2 mg L-1. Based on precision results, the LOQ was 5 mg L-1 where the RSD was 25 min. A minor interfering peak from the matrix was noted at the MT of C, which was suppressed on the electropherogram by changing the pH of the electrolyte from 7.2 to 8.0 (phosphate buffer) or 9.2 (40 mM borate buffer); however, at pH 9.2, a small interference was noted at the MT of DA. A pH of 8.0 was selected for the subsequent stages of method development, which gave reasonable MTs (10.7 and 11.25 min for DA and C, respectively) and acceptable resolution and specificity vis-a`-vis the endogenous components of the matrix. The influence of different separation voltages (10, 15, and 20 kV) and buffer concentrations (10, 20, 30, and 40 mM) at this pH was investigated, and results showed that a voltage of
15 kV and a 30 mM phosphate concentration were the best compromise for resolution, MT, and current generated. Optimization of the Peak Shape. Injection times of 3 or 5 s of undiluted spiked plasma with the separation electrolyte at pH 7.2, 8.0, and 10.0 resulted in broad and/or split peaks for DA and/or C, while no distortion of the analyte peak was observed upon injecting aqueous standard solutions at the same concentrations. The broadening of the peaks observed was related to the matrix; broad and double peaks have been previously noted for other compounds using direct injection of plasma.5 Distortion observed for plasma standards was attributed to plasma protein binding (PPB) of the drug greater than 50% and/or to a pKa value of the drug significantly lower than the buffer pH.5 These possibilities are supported by the fact that cefotaxime has a PPB around 2040%12 and pKa values of 2.1 and 4.5.7 A possible reason for the negative influence of protein binding on peak shape could be the strong interaction of acidic drugs with proteins which are slowly released from protein binding sites.5 The high ionic strength of plasma could also be partly responsible for the peak broadening or splitting; it is well established that if the sample has a high conductivity relative to the running buffer, defocusing of the solute band can take place.13 Hence, injecting a sample diluted with a solvent of low conductivity creates a high sample zone field, and as a result, sample ions migrate faster in this zone. When they reach the buffer zone where the conductivity is higher and the field lower, the rate of migration of these ions slows down, and they concentrate at the boundary. This concentration effect results in an improvement in the peak shape and, hence, in detection limits. The effect of diluting the plasma samples with water was investigated. Plasma samples spiked with C and DA (20 mg L-1 each) were injected undiluted and following dilution in water (in the proportions 3:1, 3:2, or 1:1 v/v, plasma/water), varying the injection time from 3 s (nondiluted plasma) to 4, 5, and 6 s to provide a constant amount of C injected. Dilution of plasma samples with water in the ratios 3:1 and 3:2 (v/v) resulted in significant improvement in both the peak shape and efficiency. Further dilution of plasma resulted in a slight broadening of both peaks (which can be attributed to the increase in the injection time), resulting in a lower signal-to-noise ratio. Dilution of plasma in the same ratios with a 165 mM SDS solution was also investigated as it could improve the peak shape by disrupting protein binding. In this case, however, the peak shape was not improved, possibly due to the high ionic strength of the SDS solution. As the peak shape was acceptable (Figure 3) with a dilution factor of 3:1 (v/v) plasma/water and a 3 s injection, no further investigation was carried out. Optimization of the Washing Step. A washing step with 0.1 M NaOH followed by a 2 min buffer wash was found to be effective to restore the capillary wall surface and reequilibrate the capillary between plasma injections. (ii) Performance Evaluation. Specificity. The specificity of the method vis-a`-vis endogenous components of the matrix was assessed using three different pools of human plasma. No interference was noted at the MT of C and DA (Figure 3). In addition, neither caffeine nor theophylline was found to interfere with C or DA analysis. (12) Dictionnaire Vidal; Office de vulgarisation pharmaceutique: Paris, 1995; p 319. (13) Moring, S. E.; Colburn, J. C.; Grossman, P. D.; Lauer, H. H. LC-GC Int. 1990, 3, 46-48.
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(each at five concentrations) on the same day (day 2) for cefotaxime (concentration in mg L-1) were as follows, with the confidence intervals calculated at R ) 0.05:
CPA ) (3.85 ( 0.11) × 10-4c - (0.18 ( 0.57) × 10-4; r2 ) 0.9998 RPA ) (269.24 ( 7.77) × 10-4c - (141 ( 363.63) × 10-4; r2 ) 0.9999 CPA ) (3.83 ( 0.06) × 10-4c - (2.1 ( 2.98) × 10-4; r2 ) 0.9998 RPA ) (267.10 ( 12.11) × 10-4c - (117.96 ( 566.64) × 10-4; r2 ) 0.9994 CPA ) (3.95 ( 0.16) × 10-4c + (0.97 ( 7.72) × 10-4; r2 ) 0.9995 RPA ) (268.71 ( 7.30) × 10-4c - (21.68 ( 341.60) × 10-4; r2 ) 0.9998 CPA ) (3.87 ( 0.40) × 10-4c - (3.00 ( 18.75) × 10-4; r2 ) 0.9968 RPA ) (270.35 ( 6.94) × 10-4c - (128.87 ( 102.16) × 10-4; r2 ) 0.9999 where c is concentration. Standardized residuals were randomly distributed around zero and between (2 for each graph. The RSD values between the slopes were 1.50% (CPA) and 0.50% (RPA). The results of linearity experiments carried out on four different days and calculated with the IS (see equations below) confirm that the interday precision Figure 3. Electropherograms in MECC of (a) drug-free plasma and (b) plasma spiked to 10 mg L-1 C and 5 mg L-1 DA. Table 2. Mean and RSD Values of Migration Times Obtained on Each of the Four Days in MECC (n ) 10 Injections on Days 1, 3, and 4; n ) 40 on Day 2)
day
mean MT DA (min); RSD (%)
mean relative MT DA/T; RSD (%)
mean MT C (min); RSD (%)
mean relative MT C/T; RSD (%)
1 2 3 4
10.23; 0.06 10.10; 0.36 10.12; 0.70 10.41; 0.20
0.944; 0.13 0.943; 0.04 0.943; 0.11 0.941; 0.06
10.65; 0.10 10.50; 0.35 10.55; 0.9 10.84; 0.21
0.982; 0.11 0.981; 0.02 0.980; 0.35 0.980; 0.05
RPA ) (267.56 ( 2.12) × 10-4c - (110.81 ( 98.96) × 10-4; r2 ) 0.9999 RPA ) (269.24 ( 7.77) × 10-4c - (141.90 ( 363.63) × 10-4; r2 ) 0.9998 RPA ) (260.19 ( 3.75) × 10-4c - (107.96 ( 175.49) × 10-4; r2 ) 0.9999 RPA ) (264.47 ( 15.10) × 10-4c - (427.79 ( 706.34) × 10-4; r2 ) 0.9990
Precision of Migration Times. Mean and RSD values of MTs and MTs relative to the MT of T, for both DA and C, were calculated on each of the four days (Table 2). Linearity of the Response and Precision. Optimum results were obtained using peak areas for calculations. Comparison of mean and intraday precision of the responses with and without an internal standard at each concentration shows that the use of an IS improves the precision (Table 3) because it takes into account variations in the injected volume.11,13 The equations obtained using PA/MT (CPA) and PA relative to the internal standard (RPA) for the four linearity experiments 1368
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between the slopes (RSD ) 1.49%) using an internal standard is superior to the corresponding value (RSD ) 4.38%) in the absence of an internal standard. However, the higher RSD value is still acceptable for biological applications and shows the ruggedness of MECC with direct injection of plasma even without internal standardization. For DA, the RPA relationship as a function of concentration was found to be linear in each experiment in the range investigated (r2 > 0.999), with an intraday precision at each concentration better than 8% (Table 3).
Table 3. Intraday (Day 2) Mean and RSD Values of Corrected Peak Areas (CPA) and Relative Peak Areas (RPA) at the Different Concentrations of C and DA Used in Linearity Experiments in MECC (n ) Number of Injections) analyte concn (mg L-1)
mean CPA; RSD (%)
mean RPA; RSD (%)
C5 (n ) 8) C10 (n ) 8) C20 (n ) 8) C50 (n ) 8) C100 (n ) 8) DA5 (n )16) DA10 (n ) 16) DA20 (n ) 8)
0.0170; 1.84 0.0034; 1.72 0.0071; 5.69 0.0175; 6.44 0.0355; 1.41 0.0020; 9.84 0.0043; 10.82 0.0083; 3.06
0.117; 1.41 0.235; 1.92 0.485; 1.22 1.211; 3.01 2.459; 0.31 0.126; 7.73 0.276; 5.1 0.577; 2.60
Limits of Detection (LOD) and Quantitation (LOQ). LODs were about 1 mg L-1 for C and DA (S/N ) 3), and the LOQ was estimated to be about 2 mg L-1. To determine if bound and unbound C and DA were being determined by the MECC direct injection technique, the CPAs of aqueous and plasma standards were compared at concentrations of 50 mg L-1 C + 10 mg L-1 DA + 50 mg L-1 T. The plasma CPAs as a percentage of aqueous CPAs were found to be 102.2 % (DA), 100.9% (C), and 105.9% (T). The corresponding values when T was used as internal standard were 96.5% for DA and 95.3% for C. These results demonstrate that both bound and unbound C and DA are determined using MECC with direct injection of plasma. This also indicates that aqueous standards with internal standardization could be used for the monitoring of these compounds in plasma; in addition to a better inter- and intraday precision, the internal standard allows injections to be corrected for differences of fluid viscosity which affect the injected volume. In addition, since the calibration line goes through the origin, a single calibration point is possible.
CONCLUSION CZE following acetonitrile deproteinization and MECC with direct injection of plasma samples following dilution with water are possible methods for monitoring cefotaxime and its metabolite in plasma at therapeutic levels. Both gave satisfactory results in terms of precision of migration times and linearity of the responses as a function of concentration; however, under the conditions used, precision of the slope values in CZE was inferior to the MECC method, for which a high intra- and interday precision on the slopes was obtained. Precision in the MECC procedure (even without an internal standard) is principally due to the fact that there is little sample manipulation apart from a simple aqueous dilution and there are no volatile solvents involved in the procedure. The method, without internal standardization, is sufficiently precise for the collection of reliable bioanalytical data; however, the use of an internal standard is recommended to compensate for small variations inherent in the injection system in CE. MECC allows direct injection of plasma and is, therefore, simple in its execution and yields precise results; in comparison with HPLC, the method is economical (requiring only a few milliliters of buffer and inexpensive capillaries), and the setup of the instrumentation is uncomplicated. As evidenced by the results obtained in this and other studies, MECC is proving to be an attractive alternative to HPLC in therapeutic drug monitoring and other bioanalytical applications. ACKNOWLEDGMENT The authors thank the European Community (G.C.P.) and the French Government (M.K.) for their sponsorship of this project. Received for review May 21, 1996. Accepted January 21, 1997.X AC9605049 X
Abstract published in Advance ACS Abstracts, February 15, 1997.
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