Elimination study of the chemotherapy drug tamoxifen by different advanced oxidation processes: Transformation products and toxicity assessment.

Tamoxifen is a chemotherapy drug considered as recalcitrant contaminant (with low biodegradability in conventional activated sludge wastewater treatment), bioaccumulative, ubiquitous, and potentially hazardous for the environment. This work studies the removal of Tamoxifen from water by advanced oxidation processes, paying special attention to the formation of transformation products (TPs) and to the evolution of toxicity (using the Microtox® bioassay) during the oxidation processes. Five types of treatments were evaluated combining different technologies based on ozone, hydrogen peroxide and UV radiation: i) O3, ii) O3/UV, iii) O3/H2O2 (peroxone), iv) UV and v) UV/H2O2. Complete removal of tamoxifen was achieved after 30 min for all the treatments carried out with O3 while a residual concentration (about 10% of initial one) was observed in the treatments based on UV and UV/H2O2 after 4 h of reaction. Eight TPs were tentatively identified and one (non-ionizable molecule) was suspected to be present by using ultra high performance liquid chromatography coupled to high resolution mass spectrometry. An increase of toxicity was observed during all the oxidation processes. In the case of ozone-based treatments that increase was attributed to the presence of some of the TPs identified, whereas in the case of UV-based treatments there was no clear correlation between toxicity and the identified TPs.

SiO2 nanoparticles modified CPE as a biosensor for determination of i-motif DNA/Tamoxifen interaction.

Cytosine-rich DNA sequences can form a highly ordered structure known as i-motif in slightly acidic solutions. The stability of the folded i-motif structure is a good strategy to inhibit the telomerase reaction in cancer cells. The electrochemical biosensor was prepared by modifying carbon paste electrode with SiO2 nanoparticles to investigate drugs which can stabilize this structure. Tamoxifen (Tam), an antiestrogen hormonal agent for treatment of breast cancer, was chosen as the model ligand and its interaction with i-motif structure was examined. The interaction between i-motif DNA and Tam was studied in PBS buffer and [Fe(CN)6](3-) through the cyclic voltammetry and square wave voltammetry methods. The oxidation peak of Tam, due to the i-motif DNA/Tam interaction, was observed after i-motif immobilized on the surface of the electrode. The i-motif formation was investigated by circular dichroism spectroscopy and the results showed that this structure can certainly be made with pH around 4.5, but its stability reduced by going to the more alkaline pH. The selectivity which was studied in the presence of complementary strand demonstrated that i-motif structure could be stabilized in acidic pH even in the presence of its complementary strand.

Extraction of tamoxifen and its metabolites from formalin-fixed, paraffin-embedded tissues: an innovative quantitation method using liquid chromatography and tandem mass spectrometry.

PURPOSE: Tamoxifen is a key therapeutic option for breast cancer treatment. Understanding its complex metabolism and pharmacokinetics is important for dose optimization. We examined the possibility of utilizing archival formalin-fixed paraffin-embedded (FFPE) tissue as an alternative sample source for quantification since well-annotated retrospective samples were always limited. METHODS: Six 15 µm sections of FFPE tissues were deparaffinized with xylene and purified using solid-phase extraction. Tamoxifen and its metabolites were separated and detected by liquid chromatography-tandem mass spectrometry using multiple-reaction monitoring. RESULTS: This method was linear between 0.4 and 200 ng/g for 4-hydroxy-tamoxifen and endoxifen, and 4-2,000 ng/g for tamoxifen and N-desmethyl-tamoxifen. Inter- and intra-assay precisions were <9 %, and mean accuracies ranged from 81 to 106 %. Extraction recoveries were between 83 and 88 %. The validated method was applied to FFPE tissues from two groups of patients, who received 20 mg/day of tamoxifen for >6 months, and were classified into breast tumor recurrence and non-recurrence. Our preliminary data show that levels of tamoxifen metabolites were significantly lower in patients with recurrent cancer, suggesting that inter-individual variability in tamoxifen metabolism might partly account for the development of cancer recurrence. Nevertheless, other causes such as non-compliance or stopping therapy of tamoxifen could possibly lead to the concentration differences. CONCLUSIONS: The ability to successfully study tamoxifen metabolism in such tissue samples will rapidly increase our knowledge of how tamoxifen's action, metabolism and tissue distribution contribute to breast cancer control. However, larger population studies are required to understand the underlying mechanism of tamoxifen metabolism for optimization of its treatment.

Development and validation of a quantitative assay for the determination of tamoxifen and its five main phase I metabolites in human serum using liquid chromatography coupled with tandem mass spectrometry.

A sensitive bioanalytical assay for the quantitative determination of tamoxifen and five of its phase I metabolites (N-desmethyltamoxifen, N-desmethyl-4-hydroxytamoxifen, N-desmethyl-4'-hydroxytamoxifen, 4-hydroxytamoxifen and 4'-hydroxytamoxifen) in serum is described. The method has been fully validated at ranges covering steady-state serum concentrations in patients receiving therapeutic dosages of tamoxifen. The bioanalytical assay is based on reversed phase liquid chromatography coupled with tandem mass spectrometry in the positive ion mode using multiple reaction monitoring for drug (-metabolite) quantification. The sample pretreatment consists of protein precipitation with acetonitrile using only 50 µL of serum. In the past, numerous assays have been developed by other groups for the quantification of tamoxifen and its phase I metabolites. However, the number of metabolites included in these studies is very limited and only very few of these assays have been fully validated. A liquid chromatography tandem mass spectrometry assay for the quantification of tamoxifen and four phase I metabolites in human serum that was previously developed by our group is now explicitly improved and described herein. Time of analysis has been reduced by 50% and sensitivity was increased by a reduction of the lower limit of quantification from 1.0 to 0.2 ng/mL for 4-hydroxytamoxifen and 4'-hydroxytamoxifen. Additionally, two phase I metabolites that have never been quantified in human serum hitherto, namely 4'-hydroxytamoxifen and N-desmethyl-4'-hydroxytamoxifen, were included in this assay. Validation results demonstrate an accurate and precise quantification of tamoxifen, N-desmethyltamoxifen, N-desmethyl-4-hydroxytamoxifen, N-desmethyl-4'-hydroxytamoxifen, 4-hydroxytamoxifen and 4'-hydroxytamoxifen in human serum. The applicability of the assay was demonstrated and it is now successfully used to support clinical studies in which patient-specific dose optimization is performed based on serum concentrations of tamoxifen metabolites.

Tamoxifen metabolite isomer separation and quantification by liquid chromatography-tandem mass spectrometry.

Tamoxifen (Tam), the antiestrogen used to treat estrogen receptor-positive breast cancer is a pro-drug that is converted to its major active metabolites, endoxifen and 4-hydroxy-tamoxifen (4-OH-Tam) by various biotransformation enzymes of which cytochrome P450-2D6 (CYP2D6) is key. The usual Tam dose is 20 mg daily; however, the plasma active metabolite concentrations vary due to common genetic variants encoding the biotransformation enzymes and environmental factors (e.g., concomitant drugs) that inhibit these enzymes. Effective treatment depends on adequate Tam conversion to its active isomers. To monitor metabolite plasma levels, a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to separate and quantitate Tam, N-desmethyl-tamoxifen (ND-Tam), and tamoxifen-N-oxide (Tam-N-oxide), and the E, Z, and Z' isomers of endoxifen and 4-OH-Tam. Known standards were used to identify each metabolite/isomer. Quantitation of these metabolites in plasma was linear from 0.6 to 2000 nM. Intra- and inter-assay reproducibilities were 0.2-8.4% and 0.6-6.3%, respectively. Accuracy determined by spike experiments with known standards was 86-103%. Endoxifen, 4-OH-Tam, and their isomers were stable in fresh frozen plasma for ≥6 months. This method provides the first sensitive, specific, accurate, and reproducible quantitation of Tam and its metabolite isomers for monitoring Tam-treated breast cancer patients.

Interest of molecularly imprinted polymers in the fight against doping. Extraction of tamoxifen and its main metabolite from urine followed by high-performance liquid chromatography with UV detection.

A molecular imprinted polymer (MIP) has been synthesized in order to specifically extract tamoxifen, a nonsteroidal antiestrogen, and its metabolites from urine by solid-phase extraction (SPE) before HPLC-UV analysis. Clomiphene, a chlorinated tamoxifen analogue, was selected as template for MIP synthesis. Polymerisation was achieved by thermal polymerisation of methacrylic acid (MAA) as functional monomer, ethylene glycol dimethacrylate (EDMA) as cross-linking agent and acetonitrile as porogen. The efficient elimination of the urinary matrix has been obtained by MIP-SPE but the elution recovery of tamoxifen was initially too low ( approximately 14%). This problem has been overcome following two ways. At first, a preliminary HLB-SPE of the urine has enabled to discard endogenous salts and to percolate an organic sample through the MIP cartridge. Extraction recoveries are equal to 56 and 74% for tamoxifen and 4-hydroxytamoxifen, respectively. Then, a second MIP has been prepared with styrene and MAA as functional co-monomers. Strong pi-pi interactions occurring between phenyl groups of styrene and tamoxifen promote rebinding of the analyte by the specific sites. The enhanced hydrophobic character of the imprinted polymer has enabled the direct percolation of urine through MIP-SPE and the easy elimination of endogenous salts from urine with only one aqueous washing step. HPLC-UV analysis has confirmed high extraction recoveries (85%) for tamoxifen and its metabolite with an enrichment factor of 8. This analytical protocol can selectively detect the presence of tamoxifen metabolites in urines and be useful as a proof of doping in competitive sports.

High-speed liquid chromatography/tandem mass spectrometry using a monolithic column for high-throughput bioanalysis.

With the ever-increasing workload from a variety of in vitro and in vivo screening procedures, new analytical methodologies to perform bioanalysis in an accurate and high-throughput manner are in great demand. In this work, monolithic columns were used instead of conventional particulate HPLC columns to perform chromatographic separations. Because the pressure drop on a monolithic column was considerably lower than that on a particulate column, a high flow rate (6 mL/min) was used for a 4.6 x 50 mm monolithic column with a total backpressure of about 61 bar measured using acetonitrile/water (50:50). The capability of using a regular column length at high flow rates, combined with the extremely small dependency of separation efficiency on linear flow velocity, allowed for the generation of sufficient chromatographic resolving power in a significantly reduced runtime. As demonstrated in this work, a plasma extract of a mixture of tempazepam, tamoxifen, fenfluramine, and alprozolam were baseline separated within a total analysis time of one minute. An average peak width at half maximum of approximately one second was noted using a generic broad gradient. It was also found that the separation efficiency and signal/noise (S/N) ratios for this separation remained almost constant at flow rates of 1, 3, and 6 mL/min, respectively. The ruggedness of the separation was evaluated by injecting 600 plasma extracts containing the replicates of a standard curve of the above mixture during an overnight run. The chromatographic retention time, separation quality, peak response and sensitivity were highly reproducible throughout the run. This high-speed liquid chromatography/tandem mass spectrometry (LC/MS/MS) system has been used routinely in the authors' laboratory to support drug discovery programs.

Mutagenic potential of alpha-(N2-deoxyguanosinyl)tamoxifen lesions, the major DNA adducts detected in endometrial tissues of patients treated with tamoxifen.

Breast cancer patients treated with the antiestrogen tamoxifen (TAM) show an increased risk of developing endometrial cancer. We have recently detected TAM-DNA adducts in endometrium obtained from patients treated with TAM and identified them as trans- and cis-forms of alpha-(N2-deoxyguanosinyl)tamoxifen (dG-N2-TAM). To explore the mutagenic properties of these TAM-DNA adducts, we prepared site-specifically modified oligodeoxynucleotides containing a single isomer of dG-N2-TAM by reacting a 15-mer oligodeoxynucleotide containing a single dG (5'-TCCTCCTCGCCTCTC) with tamoxifen alpha-sulfate. These modified oligodeoxynucleotides were inserted into a single-stranded shuttle vector to investigate mutagenic specificities of the adducts in simian kidney (COS-7) cells. An epimer of dG-N2-trans-TAM showed targeted mutations ranging from 0.7 to 1.5%. The other dG-N2-trans-TAM adduct showed 9.6% G-->T transversions, accompanied by 2.8% G-->A transitions. Both dG-N2-cis-TAM adducts showed similar mutation spectra, where G-->T transversions (11-12%) predominated, along with a small number of G-->A transitions and G-->C transversions. Thus, dG-N2-TAMs are mutagenic lesions in mammalian cells. The tamoxifen-DNA adducts detected in patient endometrium may cause mutations and initiate endometrial cancer.

Optimised separation of E- and Z- isomers of tamoxifen, and its principal metabolites using reversed-phase high performance liquid chromatography.

A reversed phase isocratic high-performance liquid chromatographic method is reported in which a formal structured procedure, the solvent selectivity triangle, was applied to predict the mobile phase composition giving baseline resolution of the clinically important triphenylethylene antioestrogenic agent (Z)-tamoxifen, its principal (Z)-metabolites, and also the clinically relevant (E)-geometric isomers of tamoxifen and 4-hydroxytamoxifen. The technique of solvent selectivity triangle was used to select the optimal organic modifier parameter for use with a Hichrom ODS 1 column, to achieve baseline separation of six triphenylethylene solutes. The detection system utilised post-column ultraviolet irradiation to convert solutes into their respective photocyclisation products, followed by fluorescence detection (lambda[ex] = 254 nm, lambda[em] = 360 nm), and the low detection limit for tamoxifen in serum of 0.1 microM. The optimal mobile phase composition was determined to be methanol-acetonitrile-water-trichloroacetic acid (50:31:18.9:0.1, v/v, pH 2.9). A single stage liquid-liquid extraction method for determination of triphenylethylene drugs in serum was developed. Reproducible recoveries for the (Z)-geometric isomers of tamoxifen (84 +/- 3%) and its principal metabolites including Metabolite Y (94 +/- 3%), N-desmethyltamoxifen (94 +/- 3%) and 4-hydroxytamoxifen (92 +/- 3%) were achieved, though more variable results were obtained for their corresponding (E)-geometric isomers (71 +/- 7% and 70 +/- 10%, respectively).

Column-switching techniques in the biomedical analysis of stereoisomeric drugs: why, how and when.

The application of stereoselective chromatographic techniques to bioanalytical problems has become a routine procedure. However, this approach is not always straightforward; particularly when the separation involves chromatographic chiral stationary phases. Matrix interferences and more importantly, overlapping metabolite peaks often make direct analysis impractical. One strategy to overcome these problems is to combine two or more columns with different selectivities to produce a multi-dimensional chromatographic system. This review addresses the use of coupled column chromatography in HPLC systems including different coupling methods and the application of the resulting arrangements to bioanalytical analyses.

Identification of tamoxifen-DNA adducts formed by alpha-sulfate tamoxifen and alpha-acetoxytamoxifen.

alpha-Sulfate trans-tamoxifen and alpha-sulfate cis-tamoxifen were synthesized as proposed active metabolites of tamoxifen that react with DNA. alpha-Acetoxytamoxifen was prepared as a model-activated form to produce a reactive carbocation. Calf thymus DNA was reacted with alpha-hydroxytamoxifen or the activated forms of tamoxifen, and tamoxifen-DNA adducts were analyzed by a 32P-postlabeling method. The reactivity of alpha-sulfate trans-tamoxifen to DNA was much higher than that of alpha-hydroxytamoxifen. The formation of tamoxifen-DNA adducts induced by alpha-acetoxytamoxifen and alpha-sulfate cis-tamoxifen was 1100- and 1600-fold, respectively, higher than that of alpha-hydroxytamoxifen. Both alpha-sulfate tamoxifens and alpha-acetoxytamoxifen were highly reactive to 2'-deoxyguanosine. Four reaction products of dG-tamoxifen were isolated by HPLC and characterized by mass- and proton magnetic resonance spectroscopy. Fractions 1 and 2 that eluted first were identified as the epimers of trans form of dG-N2-tamoxifen. Fractions 3 and 4 were identified as the epimers of cis form of dG-N2-tamoxifen. When DNA was reacted with alpha-acetoxytamoxifen in vitro, three isomers of dG-N2-tamoxifen were detected: fraction 2 was the major adduct while fractions 1 and 3 were minor adducts.

Fundamental studies in reversed-phase liquid-solid extraction of basic drugs; I: Ionic interactions.

Seven basic solutes with known and controlled pKa (7.93-9.5) and log P (0.23-6.63) values have been used as test probes to study the mechanism involved in liquid-solid extraction with C2 and C18 bonded silica phases. A limited comparison has also been made with underivatized silica and CN phases. In addition to the reversed-phase mechanism, cation-exchange was shown to play a very significant role in the retention process. Various cations both organic and inorganic were assessed for their elution strength, and the ordering was similar to that for classical ion-exchange chromatography. Control of selectivity in the elution process can be achieved by varying the concentration of cation or methanol in the eluent. The C2 cartridge in combination with an aqueous ammonium acetate-methanol eluent proved to be the most versatile in that all compounds, irrespective of pKa or log P could be recovered in high yield. The optimal eluent in terms of selectivity with respect to related compounds could be predicted from the solute log P. Blocking of silanols by pre-conditioning the cartridges with cations prior to sample applications was also studied. The order of cation strengths although somewhat variable was similar to that established at the elution stage. To achieve quantitative elution with methanol or aqueous methanol solutions however, high concentrations of inorganic cations, equivalent to 1 ml of a 1 M solution were required to pre-condition a 100 mg cartridge.

Biodistribution of a novel antiestrogen (Analog II) in the mouse and rat.

The biodistribution of a novel antiestrogen Analog II was determined in the mouse and rat. The tritiated product, [3H]-Analog II was prepared by New England Nuclear and was purified by preparative chromatography using silica gel and petroleum ether/methylene chloride (80:20). The fat tissue had the highest uptake due to the hydrophobic nature of Analog II. The second highest uptake was in the mouse uterine tissue which was greater than that observed in the rat. The differences in biodistribution between the mouse and rat may partially explain the differences in biological activity of Analog II previously observed in these two animal species.

Difference in extractability of estradiol- and tamoxifen-receptor complex in the nuclei from MCF-7 cells with Nonidet P-40.

The extraction of [3H]estradiol- and [3H]tamoxifen-receptor complex in the nuclei from MCF-7 cells with the nonionic detergent Nonidet P-40 has been studied. We found that there is a striking difference in the extractability of estradiol- and tamoxifen-receptor complex from nuclei with 0.5% Nonidet P-40. The nuclear bound estradiol-receptor complex is scarcely extractable with Nonidet P-40. In contrast, almost all of the nuclear bound tamoxifen-receptor complex is extractable. The nuclear [3H]tamoxifen-receptor complex extracted in the presence of Nonidet P-40 sediments in two peaks at 7 S and 5 S. The latter sedimentation rate is the same with that of the nuclear [3H]tamoxifen-receptor complex extracted with 0.4 M KCl. The nuclear [3H]estradiol-receptor complex extracted with 0.4 M KCl sediments at 4 S. The results suggest that interaction of tamoxifen-receptor complex with chromatin is different from that of estradiol-receptor complex.

Determination of tamoxifen and metabolites in human serum by high-performance liquid chromatography with post-column fluorescence activation.

Sensitive and reproducible analyses were developed for assaying tamoxifen, monohydroxytamoxifen, N-desmethyltamoxifen, metabolite E [trans-1(4-hydroxyphenyl)1,2-diphenylbut-1-ene] and a new metabolite, metabolite Y [trans-1(4-hydroxyethoxyphenyl)-1,2-diphenylbut-1-ene] in human serum using high-performance liquid chromatography (HPLC). Three different systems were developed for specific purposes. All chromatography was performed using serum extracts made with hexane-butanol. Detection was by fluorimetry of phenanthrene derivatives formed by on-stream UV irradiation with a newly described device for post-column irradiation of the HPLC stream. This device may be of use in other HPLC systems requiring post-column photochemical reactions.