Volumetric Absorptive Microsampling as a New Biosampling Tool for Monitoring of Tamoxifen, Endoxifen, 4-OH Tamoxifen and N-Desmethyltamoxifen in Breast Cancer Patients.

INTRODUCTION: In this research, we used a volumetric absorptive microsampling (VAMS) technique to collect blood samples from the patients. A rapid and simple sample preparation method and LC-MS.MS assay was then developed and validated for the simultaneous analysis of tamoxifen and its three active metabolites. METHODS: VAMS extraction was performed in methanol by sonication-assisted extraction method for 25 min after 2 hof VAMS drying. Separation was carried out using Acquity UPLC BEH C18 column (2.1 x 100 mm; 1.7 µm), with a flow rate of 0.2 mL/min, and the mobile phase gradient of formic acid 0.1% and formic acid 0.1% in acetonitrile for 5 min. The multiple reaction monitoring (MRM) values were set at m/z 358.31>58.27 for N-desmethyltamoxifen, m/z 372.33>72.28 for tamoxifen, m/z 388.22>72.28 for 4-hydroxytamoxifen, m/z 374.25>58.25 for endoxifen, and m/z 260.26>116.12 for propranolol. RESULTS AND DISCUSSION: The lower limit of quantification value (LLOQ) was 2.50 ng/mL for tamoxifen, 2.50 ng/mL for endoxifen, 1.50 ng/mL for 4-hydroxitamoxifen, and 2.00 ng/mL for N-desmethyltamoxifen. Accuracy (%bias) and precision (%CV) were within 20% for LLOQ and 15% for other concentrations. There were no interference responses >20% of the LLOQ and 5% of the internal standard. The level of ion suppression in all analytes was less than 7%. The preparation system developed in this study successfully extracted more than 90% of analytes from the matrix with precision below 15%. Carryover was shown to be below 6% in all analytes. Stability of analytes in VAMS was demonstrated for up to 30 days, under room temperature storage in a sealed plastic bag with desiccant. This method was successfully applied to analyze tamoxifen and the metabolites level in 30 ER+ breast cancer patients.

Development and validation of an UPLC-MS/MS method for the therapeutic drug monitoring of oral anti-hormonal drugs in oncology.

A liquid chromatography-mass spectrometry assay was developed and validated for simultaneous quantification of anti-hormonal compounds abiraterone, anastrozole, bicalutamide, Δ(4)-abiraterone (D4A), N-desmethyl enzalutamide, enzalutamide, Z-endoxifen, exemestane and letrozole for the purpose of therapeutic drug monitoring (TDM). Plasma samples were prepared with protein precipitation. Analyses were performed with a triple quadrupole mass spectrometer operating in the positive and negative ion-mode. The validated assay ranges from 2 to 200 ng/mL for abiraterone, 0.2-20 ng/mL for D4A, 10-200 ng/mL for anastrozole and letrozole, 1-20 ng/mL for Z-endoxifen, 1.88-37.5 ng/mL for exemestane and 1500-30,000 ng/mL for enzalutamide, N-desmethyl enzalutamide and bicalutamide. Due to low sensitivity for exemestane, the final extract of exemestane patient samples should be concentrated prior to injection and a larger sample volume should be prepared for exemestane patient samples and QC samples to obtain adequate sensitivity. Furthermore, we observed a batch-dependent stability for abiraterone in plasma at room temperature and therefore samples should be shipped on ice. This newly validated method has been successfully applied for routine TDM of anti-hormonal drugs in cancer patients.

Innovative Strategy Based on a Novel Carbon-Black-ß-Cyclodextrin Nanocomposite for the Simultaneous Determination of the Anticancer Drug Flutamide and the Environmental Pollutant 4-Nitrophenol.

In the present work, a noncovalent and eco-friendly approach was proposed to prepare a carbon-black/ß-cyclodextrin (CB/ß-CD) nanocomposite. CB/ß-CD-nanocomposite-modified screen-printed carbon electrodes were applied for the simultaneous determination of the anticancer drug flutamide (Flut) and the environmental pollutant 4-nitrophenol (4-NP). The electrochemical performance of the proposed sensor relied on the conductivity of CB, the different binding strengths of the guests (Flut and 4-NP) to the host (ß-CD), and the different reduction potentials of the nitroaromatic compounds. Fascinatingly, the proposed sensor exhibited an excellent electrochemical performance with high sensitivity, selectivity, and reproducibility. The obtained wide linear ranges were 0.05-158.3 and 0.125-225.8 µM for Flut and 4-NP. The low detection limits of 0.016 and 0.040 µM with the higher sensitivities of 5.476 and 9.168 µA µM-1 cm-2 were achieved for the determination of Flut and 4-NP, respectively. The practical feasibility of the proposed sensor was studied in tap-water and human-serum samples.

Electromembrane extraction of gonadotropin-releasing hormone agonists from plasma and wastewater samples.

In the present study, for the first time electromembrane extraction followed by high performance liquid chromatography coupled with ultraviolet detection was optimized and validated for quantification of four gonadotropin-releasing hormone agonist anticancer peptides (alarelin, leuprolide, buserelin and triptorelin) in biological and aqueous samples. The parameters influencing electromigration were investigated and optimized. The membrane consists 95% of 1-octanol and 5% di-(2-ethylhexyl)-phosphate immobilized in the pores of a hollow fiber. A 20 V electrical field was applied to make the analytes migrate from sample solution with pH 7.0, through the supported liquid membrane into an acidic acceptor solution with pH 1.0 which was located inside the lumen of hollow fiber. Extraction recoveries in the range of 49 and 71% within 15 min extraction time were obtained in different biological matrices which resulted in preconcentration factors in the range of 82-118 and satisfactory repeatability (7.1 < RSD% < 19.8). The method offers good linearity (2.0-1000 ng/mL) with estimation of regression coefficient higher than 0.998. The procedure allows very low detection and quantitation limits of 0.2 and 0.6 ng/mL, respectively. Finally, it was applied to determination and quantification of peptides in human plasma and wastewater samples and satisfactory results were yielded.

A novel luminol chemiluminescent method catalyzed by silver/gold alloy nanoparticles for determination of anticancer drug flutamide.

It was found that silver/gold alloy nanoparticles enhance the chemiluminescence (CL) of the luminol-H2O2 system in alkaline solution. The studies of UV-Vis spectra, CL spectra, effects of concentrations luminol, hydrogen peroxide and silver/gold alloy nanoparticles solutions were carried out to explore the CL enhancement mechanism. Flutamide was found to quench the CL signals of the luminol-H2O2 reaction catalyzed by silver/gold alloy nanoparticles, which made it applicable for the determination of flutamide. Under the optimum conditions, the CL intensity is proportional to the concentration of the flutamide in solution over the range 5.0 × 10(-7) to 1.0 × 10(-4)mol L(-1). Detection limit was obtained 1.2 × 10(-8)mol L(-1)and the relative standard deviation (RSD) γ5%. This work is introduced as a new method for the determination of flutamide in commercial tablets. Box-Behnken experimental design is applied to investigate and validate the CL measurement parameters.

Biopolymeric microparticles combined with lyophilized monophase dispersions for controlled flutamide release.

Despite its short half-life, no controlled release formula of flutamide (FLT) was prepared until now. Therefore, 15 chitosan microparticle formulations were prepared for oral prolonged delivery of FLT via ionotropic gelation and emulsification-ionic gelation techniques then characterized for various parameters. FLT was successfully encapsulated into microparticles with loading capacity up to 39.98% and entrapment efficiency up to 97.16% using emulsification technique. Differential scanning calorimetry indicated that FLT was retained in a crystalline form in the microparticles prepared using ionotropic gelation whereas its crystallinity was significantly reduced using emulsification technique. Relationship between formulation variables and release behavior of FLT was explored. Chitosan microparticles prepared by ionotropic gelation showed a slower FLT release with a T(25%) of 7.9h whereas microparticles prepared by emulsification-ionic gelation under the same conditions showed a quick release profile with a T(25%) of 0.3h. Using 3 different hydrophilic carriers, immediate release FLT dispersions were prepared via lyophilization of monophase solution technique then combined with prolonged release chitosan microparticles to develop 6 controlled release formulae of FLT. A wide range of FLT release profiles were generated providing a prolonged release of drug after a suitable initial burst release.

Voltammetric quantification of tamoxifen.

The electrochemical behaviour of tamoxifen has been investigated at gold electrode by cyclic voltammetry (CV), differential pulse voltammetry (DPV) and squarewave voltammetric techniques. The dependence of current and potential on pH, concentration, scan rate, nature of solvent, surfactants and different surfactant concentration is investigated. Tamoxifen is oxidized in a single two-electron, irreversible and diffusion-controlled wave. Linear calibration plots are obtained over the concentration range 1.0-5.0 and 1.0-6.0 µgmL(-1) in 1.0 M KCl and Britton Robinson buffers (pH 2.51) respectively. The procedure has been applied to the assay of the drug in tablet form with mean percentage recoveries of 99.98%. The suggested method can be successfully applied to the determination of tamoxifen in different drug formulations.

[Expression of a novel metastasis-inducing protein human anterior gradient-2 (AGR2) in breast cancer and its clinical and prognostic significance].

OBJECTIVE: To investigate the expression of a novel metastasis-inducing protein human anterior gradient-2 (AGR2) in breast cancer and its clinical and prognostic significance. METHODS: AGR2 expression was assessed in 160 cases of breast cancer and 20 cases of benign breast diseases by immunohistochemistry using tissue chip technology. In addition the expression of ERa, PR and c-erbB-2 in breast cancer was also evaluated. Follow-up information of 5-year duration was available in 127 patients with breast cancer. Kaplan-Meier analysis and COX regression model were used to analyze the correlation between AGR2 expression and the follow-up clinical data. RESULTS: The expression of AGR2 was significantly higher in breast cancers than that in benign diseases (68.3% vs. 25.0% , P < 0.01). There was a negative correlation between AGR2 expression and the histological grade of breast cancer (P <0.05) , whereas positive correlations was found between the expression of AGR2 and ERalpha (P <0.05), and between the expression of AGR2 and PR (P <0.01). In the subgroup of ERalpha-positive breast cancer, Logistic regression model demonstrated AGR2 and TNM stage were important factors affecting lymph node metastasis (both P < 0.01). Kaplan-Meier analysis demonstrated that a positive expression of AGR2 was associated with poor overall survival and relapse-free survival (both P <0.01). Moreover, COX regression model confirmed the expression of AGR2 as an independent prognostic factor among patients with ERa-positive breast cancer (P <0.01). CONCLUSIONS: The abnormal expression of AGR2 may play a role in the pathogenesis and progression of breast cancer. The metastasis-inducing capability of AGR2 may be partly regulated through the ER pathway. Therefore, AGR2 may be a useful molecular marker for prognostication for patient with hormone-responsive breast cancer.

Assay of the synthetic estrogen fosfestrol in pharmaceutical formulations using capillary electrophoresis.

This study reports--for the first time--a capillary electrophoretic method for the determination of fosfestrol, a synthetic estrogen used in the treatment of metastatic prostate cancer. The effects of the carrier ion concentration, injected volume and applied voltage were studied and optimized. A 10 mM sodium tetraborate solution was selected as the carrier electrolyte solution, while the sample was injected hydrodynamically by applying a 20 mmHg vacuum for 1 s. The driving voltage was 30 kV and the absorbance of the analyte (peak height) was monitored at 240 nm. Under the above-mentioned conditions, the migration time of fosfestrol was 6.6 min. Linearity was achieved in the analyte range 3-150 mgL(-1) with the detection limit being 1 mgL(-1). The proposed method is adequately precise (s(r)=2.8% at 100 mgL(-1) fosfestrol, n=10) without the use of an internal standard and was applied to the determination of fosfestrol in a pharmaceutical formulation. The results obtained by the proposed method were in good agreement with those derived from the USP reference method.

Herbal composition PC-SPES for management of prostate cancer: identification of active principles.

BACKGROUND: The herbal mixture PC-SPES, used to manage advanced prostate cancer, has proven thrombogenic and highly estrogenic in clinical trials. However, attempts to identify the active compounds in PC-SPES have yielded incongruous results. Moreover, warfarin was identified in the serum of a patient taking PC-SPES who experienced a bleeding disorder. To determine the active components in PC-SPES potentially responsible for these effects, we analyzed PC-SPES lots manufactured from l996 through mid-2001. METHODS: Antineoplastic activity of PC-SPES and its individual component extracts was determined by colony-forming assays with several prostate cancer cell lines, and estrogenicity was determined by analyzing expression of an estrogen-responsive reporter gene in breast cancer cells. High-pressure liquid chromatography was used to isolate, identify, and quantify components of PC-SPES. Components were also identified by proton nuclear magnetic resonance, gas chromatography/mass spectrometry, and mass spectra analysis. RESULTS: PC-SPES lots manufactured from 1996 through mid-1999 contained the synthetic compounds indomethacin (range = 1.07-13.19 mg/g) and diethylstilbestrol (range = 107.28-159.27 micro g/g) and were two to six times more antineoplastic and up to 50 times more estrogenic than lots manufactured after the spring of 1999. In lots manufactured after mid-1999, gradual declines in the concentrations of indomethacin (from 1.56 to 0.70 mg/g), diethylstilbestrol (from 46.36 to 0.00 micro g/g), and total phytosterols (from 0.586 to 0.085 mg/g) were observed. Warfarin was identified for the first time in lots manufactured after July 1998 (range = 341-560 micro g/g). In the August 2001 lot, increases were found in concentrations of the natural products licochalcone A (from 27.6 to 289.2 micro g/g) and baicalin (from 12.5 to 38.8 mg/g). CONCLUSIONS: The phytochemical composition of PC-SPES varied by lot, and chemical analyses detected various amounts of the synthetic drugs diethylstilbestrol, indomethacin, and warfarin and several natural products. To qualify for clinical pharmacologic exploration, nutritional supplements including herbal mixtures should meet standards of quality control under the Good Manufacturing Practice system, and the manufacturers of such supplements should provide reliable analytical quality assurance.

Determination of chiral ratio of o,p-DDT and o,p-DDD pesticides on polysaccharides chiral stationary phases by HPLC under reversed-phase mode.

A simple and reliable HPLC method for the chiral resolution of o,p-DDT and o,p-DDD is described. The enantiomeric resolution of o,p-DDT and o,p-DDD has been achieved on Chiralpak AD-R, Chiralcel OD-R, and Chiralcel OJ-R chiral stationary phases. The mobile phases used were acetonitrile-water (50:50 [v/v]) and acetonitrile-2-propanol (50:50 [v/v]) at a flow rate of 1.0 mL/min. For both pesticides detection was done at 220 nm. The values for o,p-DDT of alpha and R(s) varied from 1.24 to 2.52 and from 0.80 to 2.47, respectively. The values of alpha and R(s) for o,p-DDD were 1.26 and 0.60, respectively.

LC determination of aminoglutethimide enantiomers as dansyl and fluorescamine derivatives in tablet formulations.

Determination of dansyl (AG-DNS) and fluorescamine (AG-F) derivatives of rac-aminoglutethimide in tablet formulation by HPLC has been achieved on a cellulose tris-(3,5-dimethylphenyl carbamate), known as Chiralcel OD and OD-R under normal and reversed phase columns, respectively, using a fluorescence detector (lambda(ex), 360 nm; lambda(em), 530 nm for AG-DNS derivatives; lambda(ex), 395 nm, lambda(em), 495 nm for fluorescamine derivatives (AG-F)). The best results were obtained with mobile phase ethanol:cyclohexane:methanol (95:5:2 v/v/v) for AG-DNS derivatives and acetonitrile:0.5% ortho-phosphoric acid (85:15 v/v) containing 0.26 mM 1-hexanesulfonic acid sodium salt (HSA) for AG-F, respectively. The lower limit of detection (signal to noise ratio of 3:1) were found to be 20 ng ml(-1) for each enantiomer for AG-DNS and 20.5 ng ml(-1) for each diastreoisomer for AG-F.

Novel reagents for the sensitive spectrophotometric determination of flutamide, an anticancer drug in pharmaceutical preparations.

Simple and sensitive spectrophotometric methods for the determination of flutamide (FLA) in either pure form or in its pharmaceutical preparations are described. The first method is based on the diazotisation of reduced FLA, followed by coupling with alcoholic iminodibenzyl (IDB) in acid medium to give a purple coloured product having a lambda(max) of 570 nm. In the second method, the diazotisation of reduced FLA followed by coupling with 4-amino-5-hydroxy-2,7-naphthalenedisulphonic acid monosodium salt (AHND) in a buffer medium of pH 12, gives a red coloured product having a lambda(max) of 520 nm. Common excipients used as additives in pharmaceutical preparations do not interfere in the proposed methods. Both the methods are highly reproducible and have been applied to a wide variety of pharmaceutical preparations and the results compare favourably with the reported method.

Appropriate column configurations for the rapid analysis and semipreparative purification of the radiolabeled drug flutamide by high-performance liquid chromatography.

Flutamide, marketed as Eulexin, is used for treatment of metastic prostatic carcinoma. Purity of a radiolabeled batch for metabolism studies was first determined by reversed-phase HPLC on a 5 microm, 150x4.6 mm analytical column. The separation was then scaled up to give a semipreparative column (5 microm, 250x10 mm) purification procedure. Fraction analysis was done on a short rapid analysis (5 microm, 50x3.0 mm) column. Analysis of the final product was performed on the analytical column. All columns were YMC-Pack ODS-AQ. The analytical work involved large mass injections in order to have the required amounts of radioactivity needed for accurate impurity profile determinations, and the preparative work involved masses much larger than the calculated scale-up values. Ultraviolet and radiochromatograms of the drug on the various column configurations are compared. A 95.7% recovery of product was obtained, with radiochemical purity increased from 95.0 to 99.8%.

Liquid chromatography-electrospray mass spectrometry of multicomponent peptide mixtures. Characterization of a mixture from the synthesis of the hormone goserelin.

In order to separate and characterize the target peptide and the side-product peptide compounds of a synthesis mixture of the peptide hormone goserelin, liquid chromatography coupled to high-flow electrospray ionization mass spectrometry (LC-ES-MS) has been used. Goserelin is an important drug with recognized therapeutical application for palliative treatment of prostatic and breast carcinomas. Stepwise solid-phase peptide synthesis commonly results in unwanted side-products associated with incomplete peptide chains. Consequently, this procedure requires extensive purification and characterization of the final synthesis mixture. The method of linear solvation energy relationships has been applied to optimize the proportion of organic modifier of the mobile phase used in the established LC method. On the other hand, ES-MS has allowed rapid and reliable identification of the target peptide and the other impurities present in the goserelin synthesis products.

Concentrations of tamoxifen and its major metabolites in hormone responsive and resistant breast tumours.

Patients treated with tamoxifen (TAM) for primary breast cancer often manifest de novo or acquired resistance, possibly through changes in drug metabolism. Using solid-phase extraction methods and reversed-phase high-performance liquid chromatography separations, levels of TAM and metabolites 4-hydroxytamoxifen (4OH) and desmethyltamoxifen (DMT) have been measured in plasma and tumour tissue from breast cancer patients treated with TAM for at least 3 months. Patients were categorized into those with tumours responding to TAM and those showing de novo or acquired resistance. Levels of TAM, 4OH and DMT in both plasma and tissue samples were correlated with clinical response, length of treatment and patient weight. Interesting results included accumulation of 4OH in tumour tissues over time in all patients, with significance reached in the acquired resistance group. In addition, significantly lower levels of 4OH and DMT were found in plasma taken from responding patients after 3 months of treatment when compared to non-responding patients, and a small group of ER-poor patients showed significantly lower levels of all three species in plasma when compared to other patients. Whilst not explaining TAM resistance in all cases, these differences could account for the development of resistance to TAM treatment in certain subgroups of patients.

Solid-phase extraction and high-performance liquid chromatographic determination of tamoxifen and its major metabolites in breast tumour tissues.

A sensitive (200 ng/g) and selective reversed-phase high-performance liquid chromatography separation has been developed to determine the levels of tamoxifen, 4-hydroxytamoxifen (4-OH) and desmethyltamoxifen (DMT) in tumour tissue taken from patients undergoing tamoxifen therapy. A muBondapak C18 10 microm column (30 cm x 3.8 mm I.D.) was used, with a mobile phase of methanol-1% triethylamine at pH 9 (89:11, v/v). Sample preparation was carried out using a C2 (500 mg sorbent, 3 ml reservoirs) solid-phase extraction method, and extraction efficiencies were followed in individual extracts using a [3H]TAM radiolabelled spike (10000 dpm), with a range of 60-90%. Accuracy and precision (standard deviation) as determined from tumour spiked with radioinert tamoxifen and its metabolites ranged from 83.4-92.3% (+/-23-33%) at 20 microg/g; 85.2-87.7% (+/-18-23%) at 2 microg/g; 88-101% (+/-15-50%) at 0.2 microg/g and 63-94% (+/-13-24%) at 0.02 microg/g. Results from seventy-two patients show mean values (+/-S.D.) of 174+/-203 ng/g for 4-OH; 783+/-1326 ng/g for DMT and 410+/-458 ng/g for TAM, variations reflecting heterogeneity in levels between patients. This methodology can be routinely applied to the determination of tamoxifen and its metabolites in tumour tissues from patients undergoing tamoxifen therapy.

Tamoxifen-induced DNA adducts in leucocytes of breast cancer patients.

Tamoxifen-induced DNA adducts were searched in leucocyte DNA from breast cancer patients. Total white blood cell DNA from tamoxifen-treated and control patients was analysed by 32P-postlabelling using HPLC-radioactivity detection. Rat liver DNA was used as a positive standard. In blinded analysis four of the six treated patients showed DNA adducts; none of the five controls were positive. The identity of fraction as a tamoxifen adduct was confirmed by using different chromatographic systems, each with spiked rat liver samples. The level of adducts in the treated patients was 5.5 adducts/10(9) nucleotides as compared to an apparent level of 1.9/10(9) in the controls.