Development and validation of a capillary ...

IL FARMACO 60 (2005) 85–90 http://france.elsevier.com/direct/FARMAC/

Original article

Development and validation of a capillary electrophoresis method for the determination of codeine, diphenhydramine, ephedrine and noscapine in pharmaceuticals María R. Gomez a,*, Lorena Sombra b, Roberto A. Olsina b,c, Luis D. Martínez b,c, María F. Silva b,c a

Cátedra de Control de Calidad de Medicamentos, Facultad de Química, Bioquímica y Farmacia, Chacabuco y Pedernera, Universidad Nacional de San Luis, 5700 San Luis, Argentina b Area de Química Analítica, Chacabuco y Pedernera, 5700 San Luis, Argentina c CONICET, Chacabuco y Pedernera, 5700 San Luis, Argentina Received 7 July 2004; accepted 5 November 2004 Available online 22 December 2004

Abstract The present work describes a simple, accurate and rapid method for the separation and simultaneous determination of codeine, diphenhydramine, ephedrine and noscapine present in cough–cold syrup formulations by capillary zone electrophoresis. Factors affecting the separation were the buffer pH and concentration, applied voltage, and presence of additives. Separations were carried out in less than 10 min with a 20 mM sodium tetraborate buffer, pH 8.50. The carrier electrolyte gave baseline separation with good resolution, great reproducibility and accuracy. Calibration plots were linear over at least three orders of magnitude of analyte concentrations, the lower limits of detection being within the range 0.42–1.33 µg ml–1. Detection was performed by UV absorbance at wavelengths of 205 and 250 nm. Quantification of the components in actual syrup formulations was calculated against the responses of freshly prepared external standard solutions. The method was validated and met all analysis requirements of quality assurance and quality control. The procedure was fast and reliable and commercial pharmaceuticals could be analyzed without prior sample clean-up procedure. © 2004 Elsevier SAS. All rights reserved. Keywords: Capillary zone electrophoresis; Ephedrine; Noscapine; Codeine; Diphenhydramine; Pharmaceutical quality control

1. Introduction Capillary electrophoresis (CE) is a family of versatile electroseparation method which has emerged over the past 15 years into the forefront of analytical chemistry [1–3] and have garnered considerable interest from scientific community [4–7]. One of the major conquests of CE is the recognition by regulatory authorities [8–11]. The applications of CE to the area of pharmaceutical analysis and in particular to the quality control have burgeoned in recent years. The range of options available through the different operation modes creates a versatile methodology which is readily applied to a wide range of analytes. Some applica* Corresponding author. Tel.: +54-2652-425385; fax: +54-2652-422644. E-mail address: [email protected] (M.R. Gomez). 0014-827X/$ - see front matter © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.farmac.2004.11.002

tions of CE for routine quality control are the determination of drug-related impurities, assay of main component, chiral analysis, determination of inorganic ions, determination of drug residues, among others. Cough and cold remedies are over-the-counter (OTC) products used for a great number of people. Some of the most common infections are induced by cold viruses, so there is a vast demand for sinus, cough/cold and allergy OTC medications for the relief of cold symptoms. The general formulations known as cold medicines or ointments contain several active ingredients. They are usually a combination of an analgesic, an antitussive, an antihistamine and a nasal decongestant. Determination of active compounds in syrups is complicate due to the presence of inactive ingredients such as preservatives (i.e. propylparaben), coloring and flavoring agents in the preparations.

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There have been numerous publications describing various methods for the quantification of common ingredients in syrups individually and in combination with other drugs. The chromatographic and spectrophotometric techniques are the most used techniques to perform quality control routine assays. Diphenhydramine hydrochloride (DP), bromhexine and other active compounds have been determined spectrophotometrically after derivatization with different reagents [12,13]. First-derivative absorption spectroscopy was used for the determination of both bromhexine and dextromethorphan in pharmaceutical tablets [14]. Also, a determination of bromhexine by absorption spectrophotometry and multivariate calibration using partial least-squares and hybrid linear analyses was developed [15]. Other methods, such as high performance liquid chromatography (HPLC) [16–18], preparative thin layer chromatography, and gas-liquid chromatography [19], have been applied for the simultaneous determination of common drugs in pharmaceutical preparations. An ion-pairing liquid chromatography method has also been utilized successfully in the separation of the active agents found in cough–cold pharmaceutical preparations [20]. Spectrophotometric methods may require sample derivatizations or pre-treatments which can be laborious and time consuming [12,13,21–23]. In order to achieve separation of these active ingredients in a single HPLC run, the gradient mode must be employed because of their quite different hydrophobicities. The gradient elution, however, is not suited for routine analysis as a quality control method because of relatively poor reproducibility and time-consuming analysis. CE is an effective tool for drug quality control [24,25]. It provides results with substantial advantages, not only in expediency, but also in ease of operation. Furthermore, relatively low volumes of electrolyte solution are required for the electrophoretic run. Recently, some applications of CE for the analysis of active ingredients in syrups have been developed [26–28]. The purpose of this study was to develop a simple, accurate, fast and reliable CE methodology for the separation and simultaneous determination of codeine hydrochloride (CDN), DP, ephedrine hydrochloride (EP) and noscapine hydrochloride (NS) present in cough–cold syrup formulations. The effects of pH, buffer concentration and separation modes were investigated. The method was validated following the International Conference on Harmonization (ICH) guidelines [29]. The optimized methodology was successfully applied in commercial pharmaceuticals.

tem MDQ Software. Detection was performed at 205 and 250 nm. The fused-silica capillaries were obtained from MicroSolv Technology Corporation and had the following dimensions: 67 cm total length, 50 cm effective length, 75 µm ID, 375 µm OD. The temperature of the capillary and the samples was maintained at 25 °C. The pH of the electrolyte was measured by an Orion 940 pHmeter equipped with a glass-combined electrode. 2.2. Chemicals The structure and formulae of the compounds studied are shown in Fig. 1. Codeine hydrochloride, diphenhydramine hydrochloride, ephedrine hydrochloride, noscapine hydrochloride and propylparaben were purchased from SigmaAldrich Co. (St. Louis, MO). Commercial products containing the active compounds (Bisolvon Compositum™ and Amiorel Compuesto™) were supplied from a local pharmacy and manufactured by Boehringer Ingelheim (Buenos Aires, Argentina); sodium tetraborate (Na2B4O7·10H2O) and sodium acetate (NaC2H3O2) were acquired from Mallinckrodt (St. Louis, USA). The water used in all studies was ultrahigh-quality water obtained from a Barnstead Easy pure RF compact ultrapure water system. All other reagents and solvents were of analytical grade quality. All solutions were degassed by ultrasonication (Testlab, Argentina). Running electrolytes and samples were filtered through a 0.45 µm Titan Syringe filters (Sri Inc., Eaton Town, NJ, USA). 2.3. Procedure The electrolyte solution was prepared daily and filtered through a 0.45 µm Titan Syringe filters (Sri Inc.). At the beginning of the day, the capillary was conditioned with 0.1 mol l–1 NaOH for 5 min, followed by water for 5 min, and then with running electrolyte for 10 min before sample injection. To achieve high reproducibility of migration times and to avoid solute adsorption, the capillary was washed between analyses with sodium hydroxide for 2 min, followed by water for 2 min, then equilibrated with the running buffer for 4 min.

2. Experimental 2.1. Instrumentation The employed CE system consisted of a Beckman P/ACE MDQ instrument (Beckman Instruments, Inc. Fullerton, CA) equipped with a diode array detector and a data handling system comprising an IBM personal computer and P/ACE Sys-

Fig. 1. Chemical structures of (1) codeine hydrochloride; (2) noscapine hydrochloride; (3) diphenhydramine hydrochloride; (4) ephedrine hydrochloride and (5) propylparaben.


Samples were pressure-injected at the anodic side at 0.5 psi for lengths of time within the range: 3–7 s. A constant voltage was used for all the experiments. Electroosmotic flow (EOF) determination was performed by using acetone as an EOF marker. The EOF marker was prepared by diluting 1 ml of acetone with the BGE and sonication for 5 min prior injection. Stock standard solutions containing CDN, DP, EP, NS and propylparaben (PP) were prepared in buffer at concentrations of 10–100 mg l–1. For the case of NS, it was previously dissolved in ethanol, and then diluted in buffer to obtain a final ethanol concentration of 30%. A combined standard solution containing codeine, diphenhydramine, ephedrine, noscapine and propylparaben was prepared by accurately mixing the standard solutions of each drug and the resultant solution was made up to 100 ml. Diluted solutions of the commercial formulations were prepared as follows: a 10 ml aliquot of the syrups was carefully measured into a volumetric flask and diluted to 100 ml with buffer solution. The solutions were mixed and filtered through a 0.45 µm membrane.

3. Results and discussion 3.1. Method development

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Table 1 Effects of pH on the migration times and electrophoretic mobilities (µe)

EP DP CDN NS PP

tm (min) 3.371 3.587 3.803 4.017 5.186

pH 6.0 µe (cm2 V–1 s–1) 2.80 × 10–4 2.40 × 10–4 1.96 × 10–4 1.57 × 10–4 0.00

tm (min) 3.326 3.671 4.179 4.363 9.579

pH 8.5 µe (cm2 V–1 s–1) 1.99 × 10–4 1.20 × 10–4 0.28 × 10–4 0.12 × 10–4 –3.48 × 10–4

CDN: codeine; DP: diphenhydramine; EP: ephedrine; NS: noscapine; PP: propylparaben. Conditions: capillary, 67 cm full length, 50 cm effective length, 75 µm ID, 375 µm OD; hydrodynamic injection at 0.5 psi, 5 s; 20 kV constant voltage; 25 °C, detection by UV absorbance at 205 nm.

been tested, but the one producing the best results considering selectivity, reproducibility, baseline and current performance was sodium tetraborate, pH 8.50. Keeping other parameters constant (pH 8.50, 25 kV, 25 °C), the buffer concentration was varied from 5 to 75 mM. Increases in migration times as well current were observed when the concentration of buffer increased. Resolution also increased for higher buffer concentrations, but no appreciable improvements were observed for buffer concentrations above 20 mM. So, a 20 mM sodium tetraborate buffer, pH 8.50, was selected as optimal (Fig. 2). The current met an acceptable value (25 µA) and remained constant when the study was performed under the optimal experimental conditions.

In order to propose a specific and accurate way of analysing pharmaceutical products containing CDN, DP, EP and NS, by using capillary zone electrophoresis, it is essential to find the best experimental conditions in which the analytes can be separated from each other. The optimization was performed using a synthetic mixture of PP, CDN, DP, EP and NS. Propylparaben was also studied due to the fact that it is a representative preservative for syrups. The following parameters were consecutively optimized: sample conditioning, pH, BGE composition and concentration, sample and capillary temperatures, and other electrophoretic parameters such as separation voltage, injection mode and length, etc. 3.1.1. Effect of pH The buffer pH plays an important role for improving selectivity in CE. Thus, the study of the pH effect becomes critical for closely related compounds, because it affects both the overall charges of the solute and the EOF. The effect of the buffer pH was investigated within the range of 6.0–10.0 at a fixed buffer concentration, adjusted by 0.1 mol l–1 NaOH and 0.1 mol l–1 HCl. It was found that when the pH was increased, resolution also increased, while time analysis decreased (Table 1). At pH 8.50, baseline separation was achieved. 3.1.2. Effect of buffer composition and concentration Buffer concentration has also a significant effect on the separation performance through its influence on the EOF and the current produced in the capillary. Different BGEs have

Fig. 2. Electropherogram of a diluted commercial formulation (Bisolvon Compositum™). Conditions: 20 mM sodium tetraborate buffer, pH 8.50; capillary, 67 cm full length, 50 cm effective length, 75 µm ID, 375 µm OD; hydrodynamic injection at 0.5 psi, 5 s; 25 kV constant voltage; 25 °C, detection by UV absorbance at 205 nm. Peak identification: 1, ephedrine; 2, diphenhydramine; 3, codeine; 4, noscapine and 5, propylparaben.

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Table 2 Quantitative parameters of the analysis of codeine, diphenhydramine, ephedrine and noscapine by CZE in pharmaceutical samples Analyte CDN DP EP NS

Concentration range (µg ml–1) 10–100 5–100 10–100 10–100

Correlation coefficient (r2) 0.9958 0.9986 0.9950 0.9992

Slope ± SD (95%; n = 6) 24,139 ± 219 192,765 ± 926 3123 ± 82 25,188 ± 675

Intercept ± SD (95%; n = 6) –341 ± 46 16,851 ± 678 –811.52 ± 572 –323.54 ± 38

LOQ (µg ml–1)

LOD (µg ml–1)

3.45 ± 0.6 2.23 ± 0.7 1.40 ± 0.01 4.50 ± 0.8

1.07 ± 0.08 0.71 ± 0.09 0.42 ± 0.05 1.33 ± 0.06

SD, standard deviation.

3.1.3. Separation performance Fig. 2 shows the sample solution electropherograms obtained using the optimized experimental conditions. The retention times of EP, DP, CDN and NS were found to be 3.32, 3.67, 4.17 and 4.36 min, respectively. Acetone was used as an EOF marker. These migration times did not vary to any considerable degree during and in between analyses (%R.S.D. less than 1% for the retention time of each peak). Resolution between EP and DP was 1.19, from DP to CDN was 2.36 and Table 3 Precision results Method precision Method repeatability (n = 10) R.S.D. (peak area): 1.62% R.S.D. (migration time): 0.45% System repeatability (n = 10) R.S.D. (peak area): 1.43% R.S.D. (migration time): 0.31% Intermediate precision (n = 6) R.S.D. operator (migration time): 0.80% R.S.D. capillary (migration time): 0.65% R.S.D. inter-day (peak area): 1.71% R.S.D. inter-day (migration time): 0.85%

from CDN to NS was 0.80. Ephedrine, diphenhydramine, codeine, noscapine and propylparaben were baseline separated in less than 10 min, giving separation efficiencies of up to 2,445,796 experimental electrophoretic plates (N) for PP. The number of experimental N for EP, DP, CDN, and NS were approximately 54,000, 129,600, 262,000 and 3500, respectively. 3.2. Assay of a commercial product Once the conditions for separation and quantification were established, the CE method was applied to the determination of CDN, DP, EP and NS in commercial formulations (Fig. 2). The electropherogram in Fig. 2 shows no interference between active compounds and excipients of the commercial samples. 3.3. Method validation Method validation was carried out according to the ICH Guidelines [20]. Results are shown in Tables 2–4. 3.3.1. Linearity Linearity of the method was evaluated by preparing a standard solution containing CDN 135 µg ml–1; diphenhy-

Table 4 Commercial formulation a recovery test Aliquot I CDN DP EP NS Aliquot II CDN DP EP NS Aliquot III CDN DP EP NS Aliquot IV CDN DP EP NS

Base value (µg ml–1)

Quantity added (µg ml–1)

Quantity found b (µg ml–1)

Recovery (%) c

– – – –

0 0 0 0

8.974 14.981 14.973 5.022

– – – –

8.974 14.981 14.973 5.022

10 10 0 0

18.951 24.943 – –

99.77 99.62 – –

8.974 14.981 14.973 5.022

10 0 10 5

18.99 – 24.885 10.103

100.2 – 99.15 101.62

8.974 14.981 14.973 5.022

0 10 0 5

– 24.839 – 10.007

– 98.58 – 99.7

CDN, codeine; DP, diphenhydramine; EP, ephedrine; NS, noscapine. a Diluted solution of the commercial formulation, Bisolvon™; 100 × [(found-base)/added]. b Mean value (n = 6).


dramine hydrochloride 225 µg ml–1, ephedrine hydrochloride 225 µg ml–1 and noscapine hydrochloride 75 µg ml–1 (150% of targeted level of the assay concentration). Sequential dilutions of this solution were performed to give solutions at 120%, 100%, 80% and 50% of the target assay concentration. These were injected in triplicate and the corrected peak areas used to plot calibration curves. The calibration equations were calculated by the least-squares linear regression method, and unknown concentrations were calculated by interpolation. The quantities labeled for the commercial formulations are: 0.9 mg ml–1 (CDN), 1.5 mg ml–1 (DP), 1.5 mg ml–1 (EP) and 0.5 mg (NS). 3.3.2. Sensitivity The amount of standard, which could be detected with a signal-to-noise ratio ≥3 was considered to be limit of detection (LOD). The limit of quantification (LOQ) was calculated as the analyte concentrations that give rise to peak heights with a signal-to-noise ratio of 10. LOD and LOQ were determined by injecting standard combined solution at three different level concentrations for each analyte (15, 30 and 50 µg ml–1). 3.3.3. Precision (repeatability and intermediate precision) Precision expresses the degree of scatter between a series of measurements under prescribed conditions. Precision was expressed as % relative standard deviation (%R.S.D.), which were calculated for the migration times and corrected peak areas obtained for each analyte. The method repeatability refers to the variability when the method is performed by the same analyst on the same piece of equipment over a short timescale. The repeatability of the entire analytical procedure was evaluated by performing independent determinations (n = 10) on the commercial diluted solutions for each analyte. The CE system repeatability was determined by injecting ten times (n = 10) the combined solution containing CDN, DP, EP and NS at three concentration levels. The intermediate precision relates to precision when one or several factors are changed in the method within a single laboratory. Intermediate precision was evaluated changing the following factors: the capillary, the day of operation (interday repeatability) and the operator. In the first case, the separation was repeated on capillaries from different batches and different suppliers on the same day. Inter-day repeatability was evaluated over 3 days by performing six injections each day. For evaluating the intermediate precision when the operator was changed, different analysts prepared sample and standard solutions. In all the cases, the R.S.D. values for each analyte were better than those reported in Table 2.

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value. Accuracy was evaluated by performing recovery experiments. A 50 m1 aliquot of the diluted solution of the commercial formulation was collected and divided into 10 portions of 5 ml each. The proposed method was applied to six portions and the average concentrations determined for each compound were taken as a base value. Then, known quantities of the analytes (active ingredients ranging from 50% to 150% of the percent label amount) were added to the other aliquots, and the active compounds determined following the recommended procedure. For all five analytes at the different concentration levels evaluated, the recovery values met the acceptance criteria of 100 ± 2% (Table 4). 3.3.5. Specificity Specificity is described as the ability of a method to discriminate the analyte from all potential interfering substances. Specificity of the method was investigated by both peak purity and spiking experiments with pure standard compounds. Peak purity was evaluated by means of the P/ACE System MDQ Software. There appeared to be no interference from formulation excipients or other impurities present, excipients were analyzed under the same optimized conditions. 4. Conclusion The method described was found to be applicable to simultaneous determination of active ingredients in commercial cough–cold pharmaceutical preparations. Successful separation and accurate results were obtained with the single CZE mode, and it offers certain advantages in its simplicity, cost per analysis and time saving. Accordingly, the method based on CE is suitable for simultaneous determination of heterogeneous compounds in pharmaceuticals. The active compounds were determined with high efficiency in a short period of time (less than 10 min). The sample excipients did not interfere with the proposed method. It has been validated and shows a good performance with respect to selectivity, precision, linearity and accuracy, and it offers a simple, fast, inexpensive and precise way for the determination of codeine, diphenhydramine, ephedrine and noscapine in pharmaceuticals. The application of the CE method to pharmaceutical quality control should widen this area extensively and may be considered for the routine analysis of a large number of samples. On the other hand, the key features for method validation of analytical procedures developed in a pharmaceutical laboratory using HPCE were considered. This work evaluates different characteristics for the validation process, judges suitability for the intended use and also outlines the specific aspects that should be considered for a CE methodology. Acknowledgements

3.3.4. Accuracy Accuracy expresses the closeness of agreement between the value found and the value that is accepted as a reference

This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET); Agencia

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Nacional de Promoción Científica y Tecnológica (FONCYT) (PICT-BID) and Universidad Nacional de San Luis (Argentina).

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