Methodologies for Stabilization of Pharmaceuticals in Biological Samples

The instability of small drug molecules in biological fluids causes serious bioanalytical challenges, regardless of what type of powerful analytical technology (i.e., HPLC-API [atmospheric pressure ionization]/ MS-MS) is employed for the bioanalytical assay. As described in the FDA guidance for industry,1 stability is one of the basic parameters that must be measured as part of a bioanalytical method validation. It is recommended that the stability of drug components in spiked samples be determined when establishing a bioanalytical method.

In the early drug discovery stage, extensive studies of drug stability are not usually performed due to time constraints. On the other hand, drug hits that undergo rapid degradation in plasma ex vivo may yield inaccurate pharmacokinetic properties; thus it is important to measure plasma stability in the discovery setting. In the drug development stage, in which stability evaluation is obligatory, instability of drug candidates in biological samples seriously complicates assay validation. This article provides a brief review of the general methodologies commonly utilized to stabilize drug molecules that are unstable in biological matrices.

Stability measurement

Traditional procedures for drug stability measurement in spiked specimens involve sample collection over a certain period of incubation times followed by sample preparation steps such as protein precipitation to terminate the reaction prior to instrumental determination. Previously, a simple, semiautomated procedure for screening drug stability in plasma was developed in the authors’ laboratory (Drug Metabolism and Pharmacokinetics Dept., Schering-Plough Research Institute, Kenilworth, NJ).2–4 The new procedure utilizes the direct plasma injection method based on a mixed-function column high-performance liquid chromatograph combined with tandem mass spectrometry (MS-MS). 5–9 Spiked plasma samples containing drug compounds were directly and sequentially injected into a mixed-function column for the on-line removal of proteins and other macromolecules. The drug components, including their potential degradation products, were then eluted through chromatographic separation and monitored via the tandem mass spectrometer in one analytical procedure. Drug stability in plasma indicated by the change of mass chromatographic peak area for the test compounds was observed to be a function of animal species, time, and temperature. The analytical results of the drug stability experiments obtained by the semiautomated direct plasma injection method were found to correlate with those obtained by the traditional manual method using the protein precipitation procedure. This higher-throughput procedure also allows plasma stability structure relationships (PSSR) to be performed as part of lead optimization.

Strategies for stabilization

Figure 1- Flow chart of stabilization of small molecules in biological fluids.

Instability of drug molecules in biological samples is more common than that in stock solution or extract after sample processing. Biological samples are much more complex in composition than stock solutions. Thus, drug molecules that readily degrade in biological samples may still be stable in stock solution. A major cause of analyte instability in biological samples is due to enzyme activity. In plasma, the most prominent enzymatic activities are the esterase activity for compounds containing an ester group, and the deamidase activity for compounds containing an amide group.10 Esterases are a heterogeneous family of enzymes that catalyze the hydrolysis of esters and amides to their corresponding carboxylic acids. Carboxylesterases and, to a lesser extent, cholinesterases are important members of the esterase family responsible for the metabolism of drug compounds. As a result, although many ester or amide bonds are stable in solution at neutral pH, they may be susceptible to hydrolysis in plasma. The highest esterase activity is usually in the liver, but it can also be found in other tissues such as kidney, brain, red blood cells, and plasma. Drug stability in plasma may vary from different animal species, depending on the distribution of esterases in the body. Therefore, drug stability is one of the major concerns for the interpretation of drug concentrations in biological fluids. A general strategy for the stabilization of pharmaceuticals in biological samples is outlined in Figure 1.

Temperature control

The most universal approach to stabilizing drug components is to lower the temperature. Reduction of the temperature normally slows down not only enzymatic but also spontaneous reactions. For spontaneous degradation reactions, the effectiveness of temperature control depends on the activation energy. With an activation energy of 20 kcal/mol, the reaction rate is expected to be 10 times slower when the temperature is decreased from 22 °C to 0 °C (Arrhenius equation). As an example, anthracenedione, an antitumor reagent, was found to be unstable in plasma, degrading in an apparent first-order reaction with t1⁄2 about one day at room temperature. When refrigerated at 4 °C, the t1⁄2 of anthracenedione was prolonged to six days.11 For enzymatic reactions, however, the effectiveness of this approach depends on the magnitude of reduction in enzyme activity at lower temperature.

Temperature control also has implications for long-term plasma storage in the frozen state. Using NMR spectroscopy, it was demonstrated that there is a 20-fold decrease in the amount of unfrozen water in frozen human plasma when the temperature is lowered from –20 °C to –80 °C.11 As a consequence, the epimerization kinetics of moxalactam was observed to be reduced with the smaller amount of unfrozen water.12 Cisplatin, another anticancer drug for the treatment of solid tumors, and its monohydrated complex are not stable at –25 °C but are stable at –70 °C for at least three weeks.13

pH adjustment

The second approach, pH control, takes advantage of the fact that most enzymes have a narrow range of working pH. Albumin possessing weak hydrolase activity in its IIIA subdomain is the most abundant protein in animal plasma. It binds to a number of drug molecules with various affinities, conferring stability to certain compounds that are otherwise unstable in plasma. Fura et al.14 reported that the pH of ex vivo plasma, bile, and urine changes during long-term storage and sample preparation procedures such as protein precipitation, centrifugation, ultrafiltration, and evaporation. The pH shifts in biological species will significantly affect protein-binding measurement and alter the disappearance rate for pH-sensitive compounds such as acylglucuronides. 15 The pH may have a strong impact on both acid and base catalyzed enzymatic and nonenzymatic reaction.16 The addition of a small amount of appropriate buffers such as phosphate, citrate, and bicarbonate into biological samples to maintain an optimum pH was shown to be an effective way to prevent degradation.14,17 One complication for pH adjustment is that at very acidic pH, plasma proteins may precipitate, causing problems for sample storage and preparation.

Derivatization

One of the analytical purposes for the derivatization of pharmaceuticals is to ease the stability issue in biological samples. For example, drug molecules containing a sulfhydryl group (the thiol compound) are generally not stable in plasma. The thiol group is a strong nucleophile that may react with cystine residues in plasma protein or glutathione to form disulfide bonds, depending on the solution pH and oxidation potential. Derivatization is one of the useful approaches employed to stabilize this class of compounds. In this approach, the thiol compound reacts with an alkylating agent to form Michael addition derivatives.18–20 Both methyl acrylate (MA) and Nethylmaleimide have been used as the derivatizing agent. Derivatization with methyl acrylate has the advantage over that with N-ethylmaleimide, which creates a new chiral center to yield two diastereometric derivatives. As an example, Jemal and co-workers reported work on the simultaneous determination of omapatrilat and its four circulating metabolites.19Omapatrilat has a free thiol and therefore is extremely unstable in human blood and plasma at room temperature. Two of omapatrilat’s metabolites also contain a thiol and have similar plasma stability profiles. The reaction between MA and omapatrilat in plasma was conducted on ice to reduce the loss of analytes during sample processing. The resulting MA–omapatrilat products in human plasma were stable for at least 6 hr at room temperature and for more than 24 hr at 4 °C. The derivatives in plasma were then extracted for analysis.