Development and validation of a micellar liquid chromatographic method to determine three antitumorals in plasma.

AIM: A micellar liquid chromatographic method to determine several anticancer drugs (pazopanib, dabrafenib and regorafenib) in plasma was developed and validated by the guidelines of the EMA. EXPERIMENTAL: Plasma samples were directly injected, after a 1/5-dilution in a micellar solution. The drugs were resolved in <18 min using a C18 column. The mobile phase was an aqueous solution of 0.12 M SDS - 2% 1-pentanol, buffered at pH 7. The detection was performed by absorbance at 260 nm. RESULTS: The values of the main validation parameters were: LOD (0.1-1 mg/l), calibration range (0.2-2 to 80 mg/l), accuracy (-12.5 to +11.7%) and precision (<11.9%). CONCLUSION: The procedure was conducted by minimum cost, effort, manipulation, time and quantity of hazardous chemicals. The method was useful to determine the drugs at their respective target concentrations, and was found useful for clinical analysis.

A method to quantify several tyrosine kinase inhibitors in plasma by micellar liquid chromatography and validation according to the European Medicines Agency guidelines.

A procedure based on micellar liquid chromatography has been developed to monitor five tyrosine kinase inhibitors in plasma, prescribed against several kinds of cancer: erlotinib, imatinib, sunitinib, sorafenib and lapatinib. The sample was diluted in a micellar solution and directly injected, thus clean-up steps were not required. The analytes were resolved without interferences in <20 min using a C18 column and a mobile phase of 0.13 M SDS-4% 1-butanol, buffered at pH 3.5, running under isocratic mode at 1 mL/min. The detection was performed by UV-visible absorbance, using a wavelength program to maximize the signal-to-noise ratio. The method was validated following the guideline of the European Medicines Agency in terms of: selectivity, calibration range (0.05-5 µg/mol), linearity (r(2)>0.990), limit of detection (15-35 ng/mL), carry-over effect, accuracy (-10.4 to +11.0%), precision (<9.2%), matrix effect, robustness (<8.4%) and stability. The procedure is rapid, easy-to-handle, uses a low amount of toxic chemical provide reliable results. Finally, the method was successfully used to analyze the studied tyrosine kinase inhibitors in plasma from cancer patients.

Determination of tamoxifen and its main metabolites in plasma samples from breast cancer patients by micellar liquid chromatography.

A method was developed for the analysis of tamoxifen and its main derivatives (4-hydroxytamoxifen, N-desmethyl-tamoxifen, tamoxifen-N-oxide and endoxifen) in human plasma, using micellar liquid chromatography coupled with fluorescence detection. Analytes were off-line derivatized by sample UV-irradiation for 20 min to form the photocycled fluorescent derivatives. Then samples were diluted, filtered and directly injected, thus avoiding extraction steps. The analytes were resolved using a mobile phase containing 0.08 M SDS-4.5% butanol at pH 3 running at 1.5 mL/min through a C18 column at 40°C, without interferences from endogenous compounds in plasma. Excitation and emission wavelengths were 260 and 380 nm, respectively. The chromatographic analysis time was less than 40 min. The analytical methodology was validated following the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines in terms of: selectivity, linear range (0.3-15 µg/mL), linearity (r(2)>0.999), sensitivity (LOD, 65-80 ng/mL; LOQ, 165-200 ng/mL), intra- and interday accuracy (-12.2-11.5%) and precision (<9.2%) and robustness (<6.3%). The method was used to quantify the tamoxifen and tamoxifen derivatives in several breast cancer patients from a local hospital, in order to study the correlation between the genotype of the patient and the ability to metabolize tamoxifen.

Adjusting the dose of tamoxifen in patients with early breast cancer and CYP2D6 poor metabolizer phenotype.

BACKGROUND: CYP2D6 is a key enzyme in tamoxifen metabolism, transforming it into its main active metabolite, endoxifen. Poor CYP2D6 metabolizers (PM) have lower endoxifen plasma concentrations and possibly benefit less from treatment with tamoxifen. We evaluated tamoxifen dose adjustment in CYP2D6 PM patients in order to obtain plasma concentrations of endoxifen comparable to patients with extensive CYP2D6 metabolism (EM). PATIENTS AND METHODS: Comprehensive CYP2D6 genotyping and plasma tamoxifen metabolite concentrations were performed among 249 breast cancer patients in adjuvant treatment with tamoxifen. Tamoxifen dose was increased in PM patients to 40 mg and to 60 mg daily for a 4-month period each, repeating tamoxifen metabolite measurements on completion of each dose increase. We compared the endoxifen levels between EM and PM patients, and among the PM patients at each dose level of tamoxifen (20, 40 and 60 mg). RESULTS: Eleven PM patients (4.7%) were identified. The mean baseline endoxifen concentration in EM patients (11.30 ng/ml) was higher compared to the PM patients (2.33 ng/ml; p < 0.001). In relation to the 20 mg dose, increasing the tamoxifen dose to 40 and 60 mg in PM patients significantly raised the endoxifen concentration to 8.38 ng/ml (OR 3.59; p = 0.013) and to 9.30 ng/ml (OR 3.99; p = 0.007), respectively. These concentrations were comparable to those observed in EM patients receiving 20 mg of tamoxifen (p = 0.13 and p = 0.64, respectively). CONCLUSION: In CYP2D6 PM patients, increasing the standard tamoxifen dose two-fold or three-fold raises endoxifen concentrations to levels similar to those of patients with EM phenotype.

Influence of CYP2D6 polymorphisms on serum levels of tamoxifen metabolites in Spanish women with breast cancer.

BACKGROUND: Estrogen receptor-positive breast cancer tumors depend on estrogen signaling for their growth and replication and can be treated by anti-estrogen therapy with tamoxifen. Polymorphisms of the CYP2D6 and CYP2C19 genes are associated with an impaired response to tamoxifen. The study objective was to investigate the impact of genetic polymorphisms in CYP2D6 and CYP2C19 on the pharmacokinetics of tamoxifen and its metabolites in Spanish women with estrogen receptor-positive breast cancer who were candidates for tamoxifen therapy. METHODS: We studied 90 women with estrogen receptor-positive breast cancer, using the AmpliChip CYP450 test to determine CYP2D6 and CYP2C19 gene variants. Plasma levels of tamoxifen and its metabolites were quantified by high-performance liquid chromatography. RESULTS: The CYP2D6 phenotype was extensive metabolizer in 80%, intermediate metabolizer in 12.2%, ultra-rapid metabolizer in 2.2%, and poor metabolizer in 5.6% of patients, and the allele frequency was 35.0% for allele (*)1, 21.0% for *2, and 18.9% for *4. All poor metabolizers in this series were *4/*4, and their endoxifen and 4-hydroxy tamoxifen levels were 25% lower than those of extensive metabolizers. CYP2C19*2 allele, which has been related to breast cancer outcomes, was detected in 15.6% of the studied alleles. CONCLUSION: CYP2D6*4/*4 genotype was inversely associated with 4-hydroxy tamoxifen and endoxifen levels. According to these results, CYP2D6 and CYP2C19 genotyping appears advisable before the prescription of tamoxifen therapy.

Monitoring disopyramide, lidocaine, and quinidine by micellar liquid chromatography.

A micellar liquid chromatography (MLC) method using a C18 column was developed to determine three antiarrhythmic drugs--disopyramide, lidocaine, and quinidine--that are most usually monitored in serum samples. After the application of an interpretative strategy for optimization of sodium dodecyl sulfate (SDS) and modifier concentrations in order to ensure the minimum analysis time, maximum sensitivity, and good resolution, the optimum chromatographic conditions for the determination of the three antiarrhythmics were flow rate, 1 mL/min; injection volume, 20 microL; separation temperature, 25 degrees C; mobile phase, 150 mmol/L SDS-7% (v/v) butanol-phosphate buffer, 10 mmol/L, pH 7-0.9% (w/v) NaCl; and detection at 214 nm. The calibration curves for the drugs were linear (r2 > 0.999). The intraday and interday precisions were lower than 3.9% (CV). Recoveries were 100 +/- 0.6% when the method was applied to both serum samples spiked with the antiarrhythmics (n = 10) and real serum samples. In all cases, the results were similar to those obtained using the reference method (fluorescence polarization immunoassay) usually used in the Spanish hospital. The proposed method is useful for hospital monitoring of the antiarrhythmics by direct injection into the chromatograph.

Tamoxifen monitoring studies in breast cancer patients by micellar liquid chromatography.

A simple micellar liquid chromatographic procedure is described to determine tamoxifen in plasma. To perform the analysis, tamoxifen solutions were diluted in water and UV-irradiated for 20 min to form the photocycled derivative with a phenanthrene core which shows intense fluorescence. Samples were then directly injected, thus avoiding long extraction and experimental procedures. The resolution from the matrix was performed with a mobile phase containing 0.15 M SDS-7% n-butanol at pH 3 running at 1.5 mL/min through a C18 column at 40 degrees C. Detection was carried out by fluorescence, and the excitation and emission wavelengths were 260 and 380 nm, respectively. The chromatographic analysis time was less than 15 min. The analytical methodology was validated following the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines. The response of the drug in plasma was linear and in the 0.5-15 microg/mL range, with r(2) > 0.999. Accuracy and precision were <9% in both cases. The limits of detection and quantification (in nanograms per millilitre) were 50 and 150 in plasma, respectively. The method developed herein shows no interferences by endogenous compounds. Finally, the analytical method was used to determine the amount of tamoxifen in the plasma of several breast cancer patients from a local hospital.