Introduction

Since it was found few decades ago that optimization of serum antiepileptic drug levels reduced the number of seizures and drug side effects, the field of therapeutic drug monitoring (TDM) has flourished. Historically TDM started with the spectrophotometric analysis of phenobarbital and phenytoin. However, due to the inability to use spectral data to differentiate closely related drugs, chromatographic techniques such as gas chromatography (GC) and high-performance liquid chromatography (HPLC) were introduced. These methods required skill, sample preparation, and a long turn-around time (TAT) from receipt of sample to reporting of results. Thus despite their high cost, immunoassays, which provided quicker TAT, have slowly replaced chromatographic methods. Although easier to perform and suited for automation, immunoassays are usually not available when a new drug is first released to use in the treatment of patients.

Capillary electrophoresis (CE) is a very versatile technique and can potentially be used to analyze not only the drug but also its metabolites. In fact, drug analysis represents one of the best potential applications of CE. However, as with any analytical method, CE has not only advantages but also drawbacks. The advantages and disadvantages of CE and the types of drugs suited for analysis by this technique are discussed in this chapter. In general, CE offers speed, ease of analysis, low cost of operation, and very high resolution. CE can also offer basic information on the physicochemical properties of the drug such as protein binding and ionization. However, it suffers from matrix effects (especially when using serum), poor detection limits, and less than desirable precision. To use CE successfully for the

From: Clinical and Forensic Applications of Capillary Electrophoresis Edited by: J. R. Petersen and A. A. Mohammad © Humana Press Inc., Totowa, NJ

analysis of drugs, four areas require careful attention: 1) choice of the separation type (CZE, MEKC, Chiral); 2) sample preparation (especially critical for serum samples); 3) instrument setup (optimum voltage, maximum injection volume); and 4) precision (choice of capillary wash and internal standards). These points will be discussed in this chapter.

2. COMPARISON OF CE AND HPLC

In the past, small molecules, such as drugs, have not been analyzed by electrophoretic techniques due to the lack of sensitivity. Generally, drugs were analyzed by chromatographic techniques, such as HPLC, that are based on the interaction of the compound with the column packing (e.g., hydro-phobicity). CE, on the other hand, utilizes charge (directly or indirectly) to separate and identify the drug of interest.

Several studies have shown that for TDM, CE is better than HPLC, being faster and easier to use (1-6). CE also has better resolution (7), especially for polar compounds (5), and costs less to operate (7). Wynia et al. (6) determined the precision, linearity, ruggedness, and detection limits for CE and HPLC using the antidepressant drug mirtazapine. They found that the relative standard deviation (RSD) for CE was higher than that for HPLC, 0.6 vs 0.2, respectively. The linearity for CE (10-1400 ^g/mL) was also different than HPLC (4-800 ^g/mL). Altria and Bestford (8) reviewed the analysis of a variety of pharmaceuticals and found that CE has many advantages over HPLC. Many researchers have also found that CE has advantages in terms of reduced sample pretreatment, consumable costs, and analysis time. Additionally, CE has the ability to separate a wide range of compounds using a single set of operating conditions. Most workers however, agree that in general HPLC tends to give better precision and better sensitivity.

Because CE and HPLC can analyze the same molecules, in many instances they can seem complementary to each other. CE is better suited for the analysis of polar or charged compounds with high molar absorbitivity or those that are present in a relatively high concentration. Nonpolar compounds or those with low molar absorbitivity may be better analyzed by HPLC. However, by using a combination of stacking methods and special flow cells with extended light path, CE can achieve sensitivity close to that found for HPLC.

3. CE PROBLEMS FOR TDM

The poor sensitivity and matrix effects are two major problems that may seem insurmountable to newcomers to the field of CE. This is especially true for TDM. In addition, CE also has less than desirable reproducibility. These problems and how to overcome them are discussed later.

3.1. Sensitivity

Owing to the narrow path length of the detection window in the capillary, the absorbance signal in CE is not very strong. Sometimes in order to improve the separation efficiency (theoretical plate number) or to speed up the analysis, the capillary diameter is decreased. This in turn causes a further decrease in the signal. To overcome this problem, capillaries with an extended light path, e.g., Z-cell, bubble cell, or the high sensitivity cell, have been developed for some CE instruments. Even with these new detection windows, many of the drugs routinely analyzed for TDM are present in serum at concentration of 0.01-1 mg/L, well below the detection limits. Thus, the majority of the drugs have to be concentrated before analysis by CE, either on or outside the capillary. Fortunately electrophoretic techniques offer a very simple means called stacking to concentrate the analyte directly on the capillary.

Initial studies with two commonly used drugs, theophylline and phenobarbital, have demonstrated that detection of therapeutic levels are attainable with simple stacking methods and little sample preparation. Some drugs, however, still require complex extraction and concentration steps.

32. Matrix Effects

In CE, unlike most other techniques, many basic parameters such as resolution, plate number, migration time, and precision are greatly affected by the sample matrix, especially when inorganic ions or proteins are present. As the sample size increases, matrix effects become more significant (9). Generally CE easily analyzes pure standards. However, analysis of serum samples, the usual sample matrix for TDM is more difficult. The salts present in serum (~150 mmol/L) affect the field strength and consequently the velocity of the analyte causing band broadening. Proteins in the serum (~60 g/L), on the other hand, bind to the capillary walls (9), producing secondary interactions, and affecting the reproducibility of the method.

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