Optimization by factorial design of a capillary zone electrophoresis method for the simultaneous separation of antihistamines

ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 352 (2006) 41–49 www.elsevier.com/locate/yabio

Optimization by factorial design of a capillary zone electrophoresis method for the simultaneous separation of antihistamines M. Elisa Capella-Peiro´ a

a,*

, Alessandra Bossi a, Josep Esteve-Romero

b

Dipartimento Scientifico e Tecnologico, Universita` degli Studi di Verona, Strada Le Grazie 15, 37134 Verona, Italy b ´ Area de Quı´mica Analı´tica, Universitat Jaume I, Campus Riu Sec, 12080 Castello´, Spain Received 14 September 2005 Available online 28 February 2006

Abstract A 32 full factorial design was used to optimize the experimental conditions of a capillary zone electrophoresis method aimed at achieving simultaneous separation and quantification of the antihistamines brompheniramine, chlorpheniramine, cyproheptadine, diphenhydramine, doxylamine, hydroxyzine, and loratadine according to their therapeutic group. A statistical program, SPSS, was used to calculate the mathematical model with which to obtain the response surface. Critical parameters such as pH and applied voltage were studied to evaluate their effect on resolution and on efficiency. Optimum separation conditions were phosphate buffer pH 2.0, 5 kV, and 2 psi s1 at 214 nm. The analysis time was below 9 min and the theoretical plates were between 6000 and 63,000 N. Calibration curves were prepared for the antihistamines. The limits of detection were 4–14 ng mL1, which allow their quantification in pharmaceuticals. The RSD% of each antihistamine was fairly good. Up to seven antihistamines belonging to the antihistaminic H1-receptor group were separated in the same electropherogram. The proposed method was then applied to the determination of antihistamines in pharmaceutical, urine, and serum samples with recoveries in agreement with the stated contents. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Capillary zone electrophoresis; Antihistamines; Therapeutic group; Experimental design

Antihistamines are a class of pharmaceutical compound that act by stimulating the histamine action in the H1-receptors, antagonizing most of the smooth muscles [1]. Antihistamines are used to relieve or prevent the symptoms of hay fever and other allergies and to prevent motion sickness, nausea, vomiting, and dizziness. In patients with Parkinson’s disease, diphenhydramine may be used to decrease stiffness and tremors. In addition, since antihistamines may cause drowsiness as a side effect, some of them may be used against insomnia. Hydroxyzine is used in the treatment of nervous and emotional conditions to help control anxiety. It can also be used to relax patients before surgery [2]. Determination of antihistamines has been reported by chromatographic methods, such as high-performance

liquid chromatography [3,4], thin-layer chromatography [5], high-performance thin-layer chromatography [6–8], and micellar liquid chromatography (MLC)1 [9,10], by liquid chromatography coupled with mass spectrometry [11], or by gas chromatography with nitrogen–phosphor detection [12]. Differential pulse cathodic adsorptive stripping voltammetry [13,14], atomic absorption spectrometry, and colorimetric methods [15] have also been employed. Nowadays there is a need for analytical methods capable of performing simultaneous determination of active compounds in pharmaceuticals. As an example, the separation of dextromethorphan, diphenhydramine, and phenylephrine in various cough–cold formulations has been reported 1

*

Corresponding author. Fax: +34 964728066. E-mail address: [email protected] (M.E. Capella-Peiro´).

0003-2697/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.12.028

Abbreviations used: MLC, micellar liquid chromatography; CE, capillary electrophoresis; CZE, capillary zone electrophoresis; CRS, chromatographic resolution statistic; LOD, limit of detection.

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Capillary zone electrophoresis of antihistamines / M.E. Capella-Peiro´ et al. / Anal. Biochem. 352 (2006) 41–49

by using micellar liquid chromatography [10]. Nevertheless, analytes with similar structural and physicochemical properties might suffer poor resolution, due to peak overlapping. Strong interaction with the stationary phases may cause peak asymmetry and low separation efficiency. As an alternative to chromatography, capillary electrophoresis (CE) may be employed in pharmaceutical analysis. The versatility of CE in the analysis allowed the separation of a wide array of pharmaceutically relevant analytes varying in polarity, size, and stereochemistry [16,17]. The high efficiencies obtained in CE are well suited to complex mixtures in which the resolution of a large number of peaks in a short analysis time is desirable. Moreover, the United States Pharmacopoeia Convention in spring 1995 suggested reducing the number of reagents and materials used in pharmaceutical tests and assays that have the potential to cause harm to human health and the environment—a requisite which is wholly fulfilled by CE [18]. Capillary zone electrophoresis (CZE) in aqueous phosphate buffer has been successfully employed for the analysis of cyclizine contained in complex dosage forms, such as suppositories. The method had the required accuracy, selectivity, sensitivity, and precision, while degradation products, tested with stress studies, did not interfere with the detection of the analyte [19]. The influence of buffer pH on the electrophoretic behavior of 13 structurally related phenothiazines and determination of pKa values by CZE has been successfully investigated [20]. CE coupled with different detector systems, such as UV [18,21–26], diode array [27,28], and electrochemiluminescence detectors [29], has been reported for the analysis of antihistamines. In the present paper, the simultaneous determination of a group of seven antihistamines is proposed and the method is further applied to the determination of antihistamines in pharmaceuticals, in urine, and in serum. The antihistamines are classified on the basis of their therapeutic properties, as reported in Table 1. To develop the CE method for separation within the shortest time, a 32 full factorial design was used. In this case, a set of several experimental runs was required to build a surface response that gave information on a whole range of experimental conditions. CE results were used to fit a polynomial equation to create a model for the prediction of the separation results. The method showed good performance with respect to selectivity, linearity, and accuracy with respect to the mixture under investigation. The method might find application in routine monitoring of antihistamines in pharmaceutical formulations. Materials and methods Equipment Capillary electrophoretic work was performed with a BioFocus Capillary Electrophoresis System 2000 (BioRad Laboratories, Hercules, CA, USA), equipped with a UV-visible detector (190–700 nm range). The electropho-

retic runs were made at 25 ± 0.2 °C. The samples were loaded by hydrostatic pressure. The fused silica capillaries (75 lm ID, 375 lm OD) were from Polymicro Technologies (Phoenix, AZ, USA). The length of the capillaries used was 24 cm and the wavelength detection was 214 nm. The polyimide coating of the capillary was partially removed by burning at the point of detection and the uncovered portion of the capillary was aligned on the detector block. The capillary was conditioned initially by flushing with 0.1 M NaOH for 15 min followed by 15 min water and 15 min buffer. Between every injection the capillary was rinsed with water for 1 min and with electrolyte solution for 3 min to ensure a consistent electroosmotic flow. The signal was acquired by a PC connected to the capillary electrophoretic system through a Bio-Rad Chemstation. The pH was measured with a GK2401C pH meter equipped with a combined Ag/AgCl/glass electrode from Radiometer (Copenhagen, Denmark) and conductivity was recorded with a CDM 92 instrument from Radiometer. The vortex shaker was from Stuart Scientific (UK). Reagents The drugs that were analyzed (Table 1) were from Sigma (St. Louis, MO, USA). Stock solutions containing 100 lg mL1 of each compound were prepared in distilled water. The solutions were suitably diluted for the analysis. Buffer solutions were prepared with phosphoric acid, sodium dihydrogen phosphate, and disodium hydrogen phosphate, (Sigma–Aldrich). Distilled–deionized water (Millipore, Billerica, MA, USA) was used throughout. Sodium hydroxide (99% purity; Merck, Darmstadt, Germany) was used to prepare the capillary. Sample preparation The pharmaceuticals were presented as capsules (Durasina and Cariban), powders (Bisolgrip, Rinomicine, Ilvico, and Propalgina plus), oral solutions (Bisolvon compositum), and syrups (Lasa con codeina, Polaramine expectorante, and Atarax). For the analyses, several portions of the powders were taken and weighed, dissolved in water containing 2% methanol, and then diluted to an adequate concentration with distilled water. Aliquots of the solutions, suspensions, and syrups were diluted with water. All sample solutions were filtered into the autosampler vials through 0.45-lm nylon membranes with a diameter of 12 mm. The antihistamines were also analyzed in spiked urine and serum samples. Urine was diluted at a ratio of 1:10 with distilled water spiked with the antihistamines at concentrations of 1, 5, and 10 lg mL1 and these solutions were later injected into the capillary without any other kind of treatment. Serum samples spiked with the antihistamines were extracted with the solid-phase extraction method proposed by Coe et al. [30].

Capillary zone electrophoresis of antihistamines / M.E. Capella-Peiro´ et al. / Anal. Biochem. 352 (2006) 41–49

43

Table 1 Chemical structure and dissociation constants of the antihistamines pK1a

pK2a

Brompheniramine H1-receptor

9.3

—

Chlorpheniramine H1-receptor

9.2

—

Cyproheptadine H1-receptor Appetite stimulant Vascular headache suppressant

9.3

—

Diphenhydramine H1-receptor Antidyskinetic Antiemetic Antitussive Antivertigo agent Sedative–hypnotic

9.0

—

Doxylamine H1-receptor

4.4

9.2

Hydroxyzine H1-receptor Antianxiety agent Antiemetic Sedative–hypnotic

nf

nf

Loratadine H1-receptor Antiasthmatic

nf

nf

Compound therapeutic use

Structure

nf; not found. a From [31–33].

Computer modeling

Results and discussion

The SPSS program (SPSS Inc., Chicago, IL, USA) was used for the nonlinear regression analysis of the data and to obtain the empirical mathematical model that represents the response surface. Surface plot was produced by Surfer software, a contouring and three-dimensional surface mapping software program (RockWare Europe, Cureglia, Switzerland).

Experimental and screening designs The simultaneous separation and quantification of the analytes within the minimum analysis time and the maximum resolution and efficiency are the main objectives in the development of a capillary electrophoresis method for the determination of solutes in pharmaceuticals. Thus, to

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Capillary zone electrophoresis of antihistamines / M.E. Capella-Peiro´ et al. / Anal. Biochem. 352 (2006) 41–49

find the best electrophoretic conditions an optimization strategy might be of considerable help. The optimization strategy may be sequential or interpretative [34,35]. Sequential strategies have proved to have shortcomings when several local and/or secondary maxima exist; in fact the optimum did not always correspond to the best maximum. Thus, the interpretative strategy may be much more efficient and reliable. Moreover, the interpretative strategy allows interdependent variables to be taken into account. In the interpretative strategy the experiments are designed before the optimization process and used to fit a model that allows prediction of the parameters in a wide range of space. An interpretative strategy was chosen for use in the present work. One of the interpretative strategies is called factorial experiment and is based on varying all factors simultaneously at a limited number of factor levels. This kind of experimentation is especially important at the beginning of an experimental study, where the most influential factors, their ranges of influence, and factor interactions are not yet known. Factorial experiments allow experiments to take place over the whole range of the factor space. They show a high degree of precision in exchange for a minimum experimental effort, and they enable factor interactions to be detected.

ping of the peaks appears. In accordance with these results, 1 psi for 2 s was selected as the optimum injection condition. Buffer selected was phosphate because it is useful to study the change in the electrophoretic response over a wide range of pH. Molarity of the buffering agent was observed to have less influence on the separation and was therefore kept constant at 20 mM. We used methanol, ethanol, propanol, and acetonitrile as organic modifiers and results indicated that the addition of these modifiers always had a detrimental effect. The pH was studied over the 2.0 to 9.5 range, in which the charge of the analytes changes, as indicated by the pKs of the antihistamines shown in Table 1 and the electroosmotic flow, thus influencing the ionic mobility of the analytes. Finally, the effect of the applied voltage, explored over the 2- to 15kV range, shows that this factor also played a crucial role in the separation. Therefore, preliminary results indicate that the factors exerting the greatest effect on the responses of migration time, peak width and efficiency, and resolution were the pH of the running buffer and the voltage. Thus, the effects of pH and applied voltage on the migration behavior of the seven antihistamines were investigated using a capillary length of 24 cm with a 75 lm i.d., a temperature of 25 °C, injection at 1 psi for 2 s and 20 mM phosphate buffer.

Parameters selection Modeling the separation In the development of a CZE method there are several factors influencing the separation: capillary length and internal diameter, temperature of separation, sample injection, type of buffering species, addition of organic modifiers, running buffer pH, and applied voltage. The criteria for the selection of the optimum separation conditions included the achievement of the maximum separation of the analytes in the mixture within the minimum time; thus peak width (i.e., resolution) and migration time (i.e., mobility) were parameters of primary importance to assess the goodness of the experimental result. First of all, while keeping the capillary length constant at 24 cm with a 75-lm i.d. and the temperature of analysis at 25 °C, the effect of sample injection (measured as pressure multiplied by time) was evaluated with regard to peak efficiency. When the antihistamines were injected with a pressure of 1 psi for 1 s or 1 psi for 2 s the peak efficiencies (expressed as the number of theoretical plates; efficiency is related to how narrow the peaks are in an electropherogram and it is calculated by measuring the migration time and the peak width) were constant: 63,000 for brompheniramine, 47,000 for chlorpheniramine, 14,000 for cyproheptadine, 37,000 for diphenhydramine, 35,000 for doxylamine, 34,000 for hydroxyzine, and 11,000 for loratadine. Increasing the pressure time to 1 psi for 3 s peak efficiency decreases by half in the case of brompheniramine, chlorpheniramine, cyproheptadine, and loratadine, remaining constant for the other antihistamines. Finally, when the pressure was 1 psi for 4 s overload symptoms and overlap-

To evaluate the influence of pH and V on the separation we used a three-level full factorial design (32). The parameter settings and the design are reproduced in Table 2. The runs of the design were carried out in a randomized sequence and the migration times and peak widths were measured. To estimate the experimental error, replications of factor combinations were necessary. The center point was run three times. Varying all factors simultaneously at a limited number of factor levels, and after the calculation of the function responses, a polynomial curve was obtained. As responses, two different functions were checked: first, the sum of resolutions and, second, the chromatographic resolution statistic (CRS) function. The resolution (R) of a pair of peaks was calculated using Eq. (1) Table 2 32 Full factorial design and response obtained Run

pH

V(kV)

Rs

CRS

CRS1

1 2 3 4 5 6 7 8 9

4.7 2 2 9.5 4.7 4.7 9.5 2 9.5

15 5 2 5 2 5 2 15 15

17.80 90.91 5.00 9.80 1.93 10.99 0.11 15.87 30.06

9.01 4.68 266.97 465922.1 154.49 473090.6 193.01 8.48 7.98

0.11 0.21 3.74E-3 2.14E-6 6.47E-3 2.11E-6 5.18E-3 0.12 0.13

Capillary zone electrophoresis of antihistamines / M.E. Capella-Peiro´ et al. / Anal. Biochem. 352 (2006) 41–49

R¼

2ðt2  t1 Þ ; ðw2 þ w1 Þ

ð1Þ

where t1 and t2 are the migration times and w1 and w2 are the width at the peak base of two consecutive peaks, measured as time units. The numerator in Eq. (1) describes the separation process with regard to differential migration and the denominator expresses the dispersive processes acting against it. The total resolution (Rs) was set as response and calculated as the sum of the resolutions of the all pairs of peaks, Rs ¼

i X

45

using the SPSS program (data for the modeling are listed in Table 2) and the model obtained was: Rs ¼ 23:03  16:72pH þ 44:12V þ 2:12pH2  2:70V2  A09pHV  0:09pH2 V þ 0:31pHV2 .

ð4Þ

ð2Þ

R;

o

where i is the number of analytes. To quantify and interpret the relationships between responses and factor effects a response surface method was used. The general empirical model is a second-order polynomial, where the response y is related to the variables (factors) x as y ¼ b0 þ

k X i¼1

bi x i þ

k X 16i6j

bij xi xj þ

k X

bii x2i ;

ð3Þ

i¼1

where k is the number of variables (factors), b0 is the intercept parameter, and bi,bij, and bii are regression parameters for linear, interaction, and quadratic factor effects. The nonlinear regression analysis of the data was carried out

Fig. 1. Three-dimensional response surface of (A) resolution and (B) CRS1 as a function of pH and applied voltage.

Fig. 2. Electropherogram of antihistamine groups. (A) Antiemetic; (B) Sedative–hypnotic; (C) Antihistaminic H1-receptor. (1, brompheniramine; 2, chlorpheniramine; 3, cyproheptadine; 4, diphenhydramine; 5, doxylamine; 6, hydroxyzine; 7, loratadine).

Table 4 Repeatabilities (RSD%, n = 10) for the antihistamines at three different concentrations (lg mL1): c1 = 0.5, c2 = 5, c3 = 25 Compound

Brompheniramine Chlorpheniramine Cyproheptadine Diphenhydramine Doxylamine Hydroxyzine Loratadine

Intraday repeatability

Interday repeatability

c1

c2

c3

c1

c2

c3

2.8 4.1 2.9 0.4 1.0 1.6 3.8

5.1 5.9 1.8 3.5 2.4 2.9 7.3

3.5 5.6 1.3 0.6 2.7 6.9 2.9

3.0 4.3 3.5 2.4 3.8 5.0 6.3

5.6 6.1 2.3 5.2 4.5 3.9 8.5

3.6 6.0 4.5 4.8 3.9 7.0 4.9

0.6 2.7 9.93 7.39

2.0 199.6

2.3 1.7 1.8 2.1

3.5 2.04 2.2 2.05

2.6 1.91 1.5 1.96

1.6 1.98 1.9 1.98

2.9 4.06 2.2 3.94

3.3 3.9 2.6 4.12

194.6 2.94 9.69 7.31

Recovery obtained with the present method. Recovery obtained with the reference method [36].

9.40 14.07 4.43 3.94 5.99 5.42 6.76

a

0.998 0.998 0.992 0.992 0.999 0.997 0.998

b

13.6 ± 9.4 8.0 ± 5.7 6.3 ± 3.0 8.1 ± 7.3 23.5 ± 10.3 16.6 ± 10.5 3.7 ± 2.9

Atarax jarabe (UCB Pharma, S.A.) Ilvico sobres (Merck) Cariban caps. (Inibsa) Bisolvon compositum (Fher)

23.6 ± 1.0 15.8 ± 0.7 50.1 ± 4.4 30.2 ± 2.8 55.6 ± 1.5 9.2 ± 0.5 59.0 ± 2.5

Polaramine expectorante (Shering-Plough)

Brompheniramine Chlorpheniramine Cyproheptadine Diphenhydramine Doxylamine Hydroxyzine Loratadine

Propalgina plus (Roche)

LOD

Lasa con codeine (Lasa)

r

Durasina (Smith Kline & French)

Intercept

Rinomicine (Fardi)

per packet: phenylephrine hydrochloride (10), chlorpheniramine maleate (2), paracetamol (500), accharose and excipients per packet: phenylephrine hydrochloride (6), chlorpheniramine maleate (4), ascorbic acid (300), caffeine (30), paracetamol (400), salicylamide (200) and excipients per 5 ml syrup: phenylpropanolamine hydrochloride (50), chlorpheniramine maleate (4), saccharose and excipients per 5 ml syrup: pseudoephedrine hydrochloride (30), chlorpheniramine maleate (2), codeine phosphate (10) and excipients per packet: phenylephrine chlorhydrate (7.5), chlorpheniramine maleate (2), paracetamol (500), dextromethorphan hydrochloride (10), ascorbic acid (200), sodium cyclamate (180), saccharose (3670) sodium saccharine (20) and excipients per 5 ml syrup: pseudoephedrine sulfate (20), chlorpheniramine maleate (2), guaifenesine (100) saccharose, ethanol, sorbitol and excipients per 100 ml syrup: dichloride hydroxyzine (200), saccharose (75000) and excipients Per packet: paracetamol (325), caffeine (30), brompheniramine maleate (3) and excipients per capsule: doxylamine succinate (10), pyridoxine chlorhydrate (10) and excipients per 5 ml solution: ephedrine hydrochloride (7.5), diphenhydramine hydrochloride (7.5), bromhexine hydrochloride (2.5), codeine hydrochloride (10), ethanol and excipients Bisolgrip (Fher)

Slope

Table 5 Analysis of pharmaceutical preparations

Table 3 Calibration curve (1–20 lg mL1) and limits of detection (3-s criterion) in ng mL1 for the antihistamines Compound

1.96

Composition (mg) Pharmaceutical (laboratory)

where Ri, i+1 is the resolution between consecutive peaks, and Rav is the average resolution of all peaks, and Rmin is the minimum acceptable resolution, which has a value of 1. Considering that peak width is 0.1 min, two peaks, which appear contiguous, have a difference in time of 0.1 min. Thus, Rs calculus shows that the result is 1 (minimum acceptable separation). Ropt is the desired resolution, the optimum resolution, in this case 1.5. Rs is 1.5 if the two peaks are separated by a time of 0.05 min. tn is the migration time of the last-eluting solute and n is the number of compounds in the sample. The CRS considers the resolution of all solutes in the sample and incorporates three important aspects of the

1.5

Foundb (mg)

ð5Þ

1.92

Reference method

RSDa (%) Founda (mg)

RSDb (%)

The three-dimensional plot of total Rs, as a function of pH of the running buffer and voltage applied, is shown in Fig. 1A. The surface plot allows the whole range of conditions to be explored, including combinations that were not experimentally demonstrated, indicating that the maximum resolution area corresponds to pH 2.0 and to an applied voltage of 5 kV. Thus, these conditions were considered to be the optimum electrophoretic conditions to separate the seven antihistamines. The same optimum was achieved when a second response function is used, which is the inverse of the chromatographic resolution statistic. The CRS is a mathematical function calculated with Eq. (5), ( " # ) 2 n1 n1 X X R2i; iþ1 ðRi; iþ1  Ropt Þ tn CRS ¼ ; þ 2 2 n i¼1 ðRi; iþ1  Rmin Þ Ri; iþ1 i¼1 ðn  1ÞRav

2.1

Capillary zone electrophoresis of antihistamines / M.E. Capella-Peiro´ et al. / Anal. Biochem. 352 (2006) 41–49

CE method

46

Capillary zone electrophoresis of antihistamines / M.E. Capella-Peiro´ et al. / Anal. Biochem. 352 (2006) 41–49

separation. The first term in Eq. (5), named the resolution term, evaluates the resolution between all adjacent solute pairs in comparison to defined values for optimum and minimum resolution. The second term in Eq. (5), named the distribution term, considers the relative spacing of

47

the solute zones. The final multiplier term in Eq. (5) takes into consideration the analysis time and the number of analyte peaks to be separated. The CRS values obtained for each electrophoretic condition and also inverse of CRS are shown in Table 2.

Fig. 3. Blank (A), chlorpheniramine-spiked (C), and diphenhydramine-spiked (E) urine samples; blank (B), chlorpheniramine-spiked (D), and diphenhydramine-spiked (F) serum samples.

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Capillary zone electrophoresis of antihistamines / M.E. Capella-Peiro´ et al. / Anal. Biochem. 352 (2006) 41–49

The inverse of CRS was chosen because the maximum of the function fits the optimal condition. The response surface of this response function (inverse of CRS) was compared with the results obtained with the Rs function. Fig. 1B shows the surface plot and the maximum of this function coincided with the optimum conditions obtained with the Rs function. The mathematical model obtained was Eq. (5). CRS1 ¼ 0:16 þ 0:04pH þ 0:11V  0:001pH2  0:005V2  0:03pHV þ 0:001pH2 V þ 0:001pHV2 .

ð6Þ

To summarize, the conditions selected were as follows: a capillary length of 24 cm with a 75 lm i.d., a temperature of 25 °C, injection at 1 psi for 2 s, 20 mM phosphate buffer, pH 2.0, and a voltage of 5 kV. Under these conditions, the migration times of the compounds were 3.8, 4.2, 4.3, 6.0, 6.3, 6.8, and 8.3 for doxylamine, brompheniramine, chlorpheniramine, cyproheptadine, hydroxyzine, diphenhydramine, and loratadine, respectively. Fig. 2 shows the electropherograms of the antihistamines belonging to antiemetic, sedative–hypnotic, and antihistaminic H1-receptor categories with a good resolution under the optimum conditions. Linearity The calibration graphs for the seven antihistamines were constructed by triplicate injection of five solutions of each drug at increasing concentrations in the 0.2- to 20-lg mL1 range. Table 3 summarizes the parameters of the calibration curves obtained for each antihistamine analyzed by measuring peak areas. Linear regression coefficients were always r > 0.99. Limits of detection The LOD of a method is the lowest analyte concentration that produces a response detectable above the noise level of the system—typically three times the noise level. In all cases, the LODs were in the 4- to 14-ng mL1 range, which is well below those required for analysis of the antihistamines in the pharmaceutical preparations (Table 3). These LODs are also good for the determination of the antihistamines in biological fluids and are similar to those found in the literature [18,21–26].

Analysis of pharmaceutical, urine, and serum samples The optimized procedure was applied to the analysis of antihistamines in the pharmaceuticals listed in Table 5 and compared with a reference method consisting of micellar liquid chromatography. The recoveries were in the 96– 103% range. These results agree well with those declared by the manufacturers. The other compounds contained in the pharmaceutical preparations, such as pseudoephedrine, ascorbic acid, bromhexine, caffeine, codeine, pyridoxine, dextromethorphan, paracetamol, and salicylamide, did not interfere with the antihistamine determination. The results of the analysis indicate that the optimized electrophoretic method is well suited to the assay of the drugs contained in pharmaceuticals due to the satisfactory recoveries obtained. To demonstrate the usefulness of this procedure, blank urine samples were spiked with known amounts of the antihistamines in the 1- to 10-lg mL1 range. The urine samples were diluted 1:10 to minimize the differences in conductivity between the sample and the running electrolyte. Spiked urine samples were analyzed by the method proposed here and with the MLC method; both results were adjusted by lineal regression, and it was found that CE = 0.03 + 0.93 * MLC (r = 0.96). Spiked serum samples were also prepared and analyzed, the result being CE = 0.14 + 0.89 * MLC (r = 0.92). Fig. 3 presents the urine and serum blanks electropherogram and two spiked samples. These results show how the method proposed here is useful for determining antihistamines in urine and serum samples, bearing in mind that the method was also applied to the determination in pharmaceuticals. Conclusions This work demonstrates that the 32 full factorial design provides a suitable means of optimizing, and different antihistamines belonging to the same therapeutic group can be separated and quantified in one single injection in CZE. The experimental runs were used effectively to build a simulated separation that allows extrapolation of the optimum separation conditions. The method was simple, very sensitive, and fast. The simultaneous identification and quantification of the seven antihistamines was achieved with high repeatability and reproducibility, and thus the method might well be suitable for quality control analyses of antihistamines in pharmaceuticals, urine, and serum.

Precision Acknowledgments The intraday repeatabilities (average of 10 measurements made on the same day) and interday repeatabilities (average of 10 intraday values taken over 5 consecutive days) are indicated in Table 4 at three different drug concentrations (0.5, 2.5, and 5 lg mL1) The relative standard deviations (RSD) were below 5% for most compounds and similar to those reported in the literature.

This work was supported by the Agencia Valenciana de Ciencia i Tecnologia de la Generalitat Valenciana (Spain), which provided Maria-Elisa Capella-Peiro´ with a research grant. Alessandra Bossi thanks MIUR (PRIN 2003) for partially supporting the present work. Josep Esteve-Romero is grateful to Project Bancaixa (P1A2003-07).

Capillary zone electrophoresis of antihistamines / M.E. Capella-Peiro´ et al. / Anal. Biochem. 352 (2006) 41–49

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