Gradient RP-HPLC method for the determination of potential impurities in atazanavir sulfate

This paper proposes a simple and selective RP-HPLC method for the determination of process impurities and degradation products (degradants) of atazanavir sulfate (ATV) drug substance. Chromatographic separation was achieved on Ascentis^® Express C8, (150 mm × 4.6 mm, 2.7 μm) column thermostated at 30 °C under gradient elution by a binary mixture of potassium dihydrogen phosphate (pH 3.5, 0.02 M) and ACN at a flow rate of 1.0 ml/min. A photodiode array (PDA) detector set at 250 nm was used for detection. Stress testing (forced degradation) of ATV was carried out under acidic, alkaline, oxidative, photolytic, thermal and humidity conditions. In presence of alkali, ATV transformed into cyclized products and the order of degradation reaction is determined by the method of initial rates. The unknown process impurities and alkaline degradants are isolated by preparative LC and characterized by ESI-MS/MS, ¹H NMR, and FT-IR spectral data. The developed method is validated with respect to sensitivity (lod and loq), linearity, precision, accuracy and robustness and can be implemented for routine quality control analysis and stability testing of ATV.     

An HPLC-MS/MS method for the quantitative determination of 4-hydroxy-anethole trithione in human plasma and its application to a pharmacokinetic study

A selective, rapid and sensitive method for the quantitation of 4-hydroxy-anethole trithione (ATX) in human plasma based on high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) was developed and validated. Paracetamol was used as the internal standard (I.S.). After liquid–liquid extraction of 500 μL plasma with ethyl acetate, ATX and the I.S. were chromatographed on an Inertsil^® ODS-3 column. The mobile phase was consisted of methanol–water (75:25, v/v) with a flow rate of 0.25 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The calibration curve was linear over the range of 0.452–603 ng/mL (r² ≥ 0.99) with a lower limit of quantitation (LLOQ) of 0.452 ng/mL. The intra- and inter-day precision (relative standard deviation, R.S.D.) values were below 13% and the accuracy (relative error, R.E.) was from −2.7% to −7.5% at three quality control levels. The assay herein described was successfully applied to a pharmacokinetic study of anethole trithione (ATT) tablet in healthy volunteers after oral administration.     

Rapid identification and absence of drug tests for AG-013736 in 1mg Axitinib tablets by ion mobility spectrometry and DART™ mass spectrometry

Axitinib (AG-013736) is a potent investigational drug that has antitumor activity in patients with metastatic renal cell carcinoma and other types of cancers. In this study, ion mobility spectrometry and “direct analysis in real time” (DART™) mass spectrometry were used to rapidly identify AG-013736 in drug substance samples and 1 mg Axitinib tablets. The plasmagrams of the sample solutions exhibited a major peak with a reduced ion mobility that was within ±0.0002 cm² V⁻¹ s⁻¹ of that for AG-013736 in an external reference standard solution. The DART ionization source was coupled with both a time-of-flight mass spectrometer and a lower-resolution ion trap mass spectrometer. Samples were analyzed by this technique in as little as 5 s with minimal to no sample preparation required. The isotopic masses of the protonated dimer ions of AG-013736 were used to identify AG-013736 in the active tablet. Both techniques were also used to develop low-level limit tests for rapidly verifying the presence or absence of AG-013736 in blinded clinical supplies of active and matching placebo tablets of Axitinib.     

LC and LC–MS/MS study of forced decomposition behavior of anastrozole and establishment of validated stability-indicating analytical method for impurities estimation in low dose anastrozole tablets

Anastrozole tablets were subjected to different ICH prescribed stress conditions of thermal, hydrolysis, humidity, photolysis and oxidation stress. The drug was found to be stable for all the stressed conditions except for oxidation. Separation of anastrozole from its potential impurities, degradation products and five anastrozole related compounds as main impurities were achieved on Inertsil ODS-3V, 250 mm × 4.6 mm i.d, 5 μm analytical column using reversed phase high performance liquid chromatography (RP-HPLC). The elution of impurities employed time dependent gradient programmed mobile phase consisting of water as mobile phase-A and acetonitrile as mobile phase-B at column flow rates of 1 ml/min and at 215 nm UV detection. The same method was also extended to LC–MS/MS studies which were carried out to identify the degradation product. The method developed was established to have sufficient intermediate precision as similar separation was achieved on another instrument handled by a different operator. The LOQ for anastrozole related compound-A (RC-A), related compound-B (RC-B), related compound-C (RC-C), related compound-D (RC-D), related compound-E (RC-E) and anastrozole were 0.05, 0.03, 0.03, 0.06, 0.06 and 0.06 μg ml⁻¹ respectively. The linearity of the proposed method for all the above related compounds was investigated in the range of LOQ to 0.600 μg ml⁻¹ respectively. The specificity was established through peak purity testing using a photo-diode array detector. Method was validated according to ICH guidelines and statistical analysis of the data proved to be suitable for stability testing at quality control.     

Maternal self concept as a provider and cessation of substance use during pregnancy

Objective

Maternal substance use during pregnancy is a common modifiable risk factor for poor birth outcomes, and is associated with long term psychological risks to offspring. As self concept is known to affect substance use behaviors in non-pregnant women, we hypothesized that self concept as a provider may be particularly salient to cessation of use during pregnancy. To isolate psychological processes specific to pregnancy from those associated with the transition to parenthood, we examined birth mothers who made adoption placements participating in the Early Growth and Development Study.

Methods

We obtained lifetime and pregnancy substance use history and psychological measures at 3 to 4 months postpartum from 693 women recruited from the Northwest, Southwest, and Mid-Atlantic regions of the United States. Life history calendar and computer-assisted personal interviewing methods were used to minimize reporting bias. Using logistic regression, we assessed the association of self concept as an adequate provider with cessation of substance use during pregnancy, controlling for sociodemographic variables, depressive symptoms experienced during pregnancy, past year antisocial behaviors, family history of substance abuse, timing of pregnancy recognition, timing of initiation of prenatal care, and emotional adjustment to the adoption decision.

Results

More positive self-concept as an adequate provider was independently associated with cessation of substance use and earlier initiation of prenatal care during pregnancy [OR = 1.223; 95% C.I. (1.005–1.489); B(SE) = .201(.100)]. Familial substance abuse, depressive symptoms, and antisocial behaviors during pregnancy, were also independent predictors, and more strongly associated with cessation [OR = .531; 95% C.I. (.375–.751); B(SE) = −.634 (.178)], [OR. 940; 95% C.I. (.906–.975); B(SE) = −.062 (.019)], [OR = .961; 95% C.I. (.927–.996); B(SE) = −.040 (.018)].

Conclusions

Enhancing maternal identity as a provider for the fetus during pregnancy, along with treatment of depression, may improve motivation to stop substance use.     

Determination of platinum originating from carboplatin in canine sebum and cerumen by inductively coupled plasma mass spectrometry

We present highly sensitive, reliable methods for the determination of platinum originating from carboplatin in canine sebum and cerumen. The methods are based on the measurement of platinum by inductively coupled plasma mass spectrometry and allow quantification of 0.15 pg platinum per cm² body surface in canine sebum and of 7.50 pg platinum per sampled ear canal. The sample pretreatment procedure involved extraction of wipe samples followed by dilution with appropriate diluents. The performance of the methods, in terms of accuracy and precision, fulfilled the most recent FDA guidelines for bioanalytical method validation. Validated ranges of quantification were 15.0–1.00 × 10⁴ ng L⁻¹ for platinum in canine sebum extraction solution (corresponding to 15.0 pg per wipe sample or 0.15 pg cm⁻²) and 7.50–1.00 × 10⁴ ng L⁻¹ for platinum in canine cerumen extraction solution (corresponding to 7.50 pg per sampled external acoustic meatus). Canine matrices may not always be obtained in sufficient quantities. Therefore, we also confirmed the legitimacy of the application of human matrix samples for the preparation of calibration standards and quality control samples as alternatives, to be used in future clinical studies. The assays are used to support human biomonitoring studies and pharmacokinetic oncology studies in pet dogs treated with carboplatin.     

Optimization of separation and determination of moxifloxacin and its related substances by RP-HPLC

A RP-HPLC method for the separation and determination of impurities of moxifloxacin, in its pharmaceutical forms as well as moxifloxacin degradation products, was developed with the aid of DryLab^® software and chemometric (response surface) approach. The separation of four synthesis-related impurities was achieved on a Waters C₁₈ XTerra column using a mobile phase of (water + triethylamine (2%, v/v)): acetonitrile = 90:10 (v/v%); the pH of water phase being adjusted with phosphoric acid to 6.0. Flow rate of the mobile phase was 1.5 ml/min and UV detection at 290 nm was employed. The column was thermostated at 45 °C. The resolution between the two least resolved impurity peaks was in average, R_s,min > 1.5. Method validation parameters indicate linear dynamic range 0.2–2.0 μg/ml with LOQ ca. 0.20 μg/ml and LOD ca. 0.05 μg/ml for all analytes.     

Monitoring of cefpirome–antioxidant interaction studies using RP-HPLC

The present work is aimed to develop and validate a method for the determination of cefpirome from bulk drug and its application in cefpirome–antioxidant interactions. Separation was achieved on a Mediterranea C18 (5?μm, 25?×?0.46?mm) column, mobile phase consisting of methanol:water (30:70, v/v). Mobile phase was pumped at a flow rate of 1?ml?min^?1 using isocratic pump system and chromatograms were recorded at 265?nm. Linearity was studied up to 50?μg?ml^?1 with correlation coefficient (r ²) of 0.9999. The limits of detection (LOD) and quantification (LOQ) were 0.03 and 009?μg?ml^?1, respectively. The intraday and inter-day precision of the method was less than 2% and accuracy was between 98.85 and 100.16%. The method was applied to investigate the interaction of cefpirome with antioxidants (vitamin A, C, E, and sodium metabisulfite). These interactions were evaluated at 37°C for half an hour. The samples were then analyzed by RP-HPLC. The results showed that after interaction with antioxidants, availability of cefpirome was decreased to 58.97, 49.85, 50.51, and 50.15% in the presence of vitamin A, C, E, and sodium metabisulfite, respectively. On the basis of interaction results, it can be concluded that whenever coadministration of cefpirome is required with antioxidants, a proper interval should be given in order to avoid interactions.     

Transdermal iontophoresis of ranitidine: an opportunity in paediatric drug therapy

The objective of this study was to examine the use of transdermal iontophoresis for the delivery of ranitidine hydrochloride in children. Constant, direct current, anodal iontophoresis of ranitidine was performed in vitro across dermatomed pig skin. The effect of donor vehicle, current intensity, and drug concentration were first examined using aqueous solutions. It was found that drug delivery was higher at pH 7 (donor: 5 mM Tris) than pH 5.6 (donor: water). In the presence of low levels of competing background electrolyte, ranitidine delivery increased linearly with applied current but was independent of the donor drug concentration. The second part of the study evaluated two Pluronic^® F-127 gels as potential vehicles for ranitidine delivery. The formulations were characterised in terms of apparent viscosity, conductivity and passive permeation measurements. Iontophoretic delivery of ranitidine was only slightly affected when delivered from the gels relative to aqueous solutions. Overall the results demonstrated that therapeutic paediatric doses of ranitidine (neonates: 0.09–0.17 μmol/kg h; 1 month to 12 years: 0.36–0.71 μmol/kg h) could be easily achieved by transdermal iontophoresis with simple gel patches of practical surface area (0.2–1.5 cm²/kg).     

Rapid and sensitive simultaneous determination of ezetimibe and simvastatin from their combination drug products by monolithic silica high-performance liquid chromatographic column

A simple, precise and rapid high-performance liquid chromatography (HPLC) method has been developed and validated for the simultaneous determination of ezetimibe (EZE) and simvastatin (SIM) from their combination drug products. The applicability of monolithic LC phases in the field of quantitative analysis has been evaluated. The existing method with UV detection set at 240 nm was successfully transferred from a conventional silica column to a 10 cm × 4.6 mm i.d. monolithic silica column. By simply increasing the mobile phase flow rate, run time was about five-fold reduced and the consumption of mobile phase was about two-fold decreased, while the chromatographic resolution of the analytes remain unaffected. Ranitidine (RAN) was used as internal standard to guarantee a high level of quantitative performance. The method used a mobile phase consisted of acetonitrile–ammonium acetate (50 mM pH 5.0) (65:35, v/v). It was validated with respect to system suitability, specificity, limit of quantitation (LOQ) and detection (LOD), linearity, precision, accuracy, and recovery, respectively. The described method was linear over the range of 40–1200 ng ml⁻¹ (r = 0.999) for both drugs. The LOD for EZE and SIM were 13.2 ± 0.4029 and 13.3 ± 0.4772 ng ml⁻¹, respectively. The LOQ were found to be 39.9 ± 1.221 and 39.5 ± 1.446 ng ml⁻¹ for EZE and SIM, respectively. The method is fast (less than 2.0 min) and is suitable for high-throughput analysis of the drug and ones can analyze 700 samples per working day, facilitating the processing of large-number batch samples.     

Validation of a capillary electrophoresis method for the analysis of ibandronate related impurities

A capillary zone electrophoretic (CZE) method has been developed for the determination of impurities (phosphyte and phosphate) in technical-grade ibandronate, which is a potent nitrogen-containing bisphosphonate. Successful separation of the drug from the impurities was achieved using 1mM tetradecyl-trimethyl-ammonium bromide (TTAB) and 5mM potassium chromate (pH 10.0) as background electrolyte with an indirect detection at 254 nm. The optimised method was validated for specificity, precision, linearity and accuracy. The limit of detection (LOD) was 2 microg/mL and the limit of quantification (LOQ) was 7 microg/mL for both phosphyte and phosphate. The developed CZE method used to determine phosphyte and phosphate as bisphosphonates impurities can be used to evaluate the quality of regular production samples of ibandronate.     

A developed HPLC method for the determination of Alogliptin Benzoate and its potential impurities in bulk drug and tablets

Alogliptin (AGLT), active ingredient of Alogliptin Benzoate (AGLT-BZ), is a new dipeptidyl peptidase-4 (DPP-4) inhibitor for the treatment of type 2 diabetes. This study aimed to build a suitable method to determine the potential related substances in AGLT-BZ bulk drug and tablets. Seven related substances in Alogliptin Benzoate substances were synthetized and identified by ¹H-NMR and ESI-MS. In addition, the impurities were detected by a gradient reverse-phase high performance liquid chromatography (RP-HPLC) with UV detection. The chromatographic system consisted of an Angilent Zobax SB-CN column (250 × 4.6 mm; 5 μm). The mobile phase consisted of water/acetonitrile/trifluoroacetic acid 1900:100:1 v/v/v (solution A) and acetonitrile/water/trifluoroacetic acid 1900:100:1 v/v/v (solution B) using a gradient program at a flow rate of 1.0 ml/min with 278 nm detection and an injection volume of 20 μl. Additionally, selectivity, the limit of quantitation (LOQ) and limit of detection (LOD), linearity, accuracy, precision and robustness were determined. Linearity was good over the concentration range 50–1000 ng/ml and the coefficient of determination (R²) were 0.9991–0.9998. RSD% of the determination of precision were <2% (n = 6). The method of RP-HPLC for the determination of impurities in AGLT-BZ was proved to be precise, accurate, robust and reliable. Three batches of self-made bulk drug and three dosages of commercial tablets were detected with this method.     

Identification of new impurities of enalapril maleate on oxidation in the presence of magnesium monoperoxyphthalate

Stress stability testing and forced degradation were used to determine the stability of enalapril maleate (EM) and to find a degradation pathway for the drug. The degradation impurities, formed under different stressed conditions, were investigated by HPLC and UPLC–MS methods. HPLC analysis showed several degradation impurities of which several were already determined, but on oxidation in the presence of magnesium monoperoxyphthalate (MMPP) several impurities of EM were observed which were not yet characterized. The HPLC methods for determination of EM were validated. The linearity of HPLC method was established in the concentration range between 0.5 and 10 μg/mL with correlation coefficient greater than 0.99. The LOD of EM was 0.2 μg/mL and LOQ was 0.5 μg/mL. The validated HPLC method was used to determine the degradation impurities in samples after stress stability testing and forced degradation of EM. In order to identify new degradation impurities of EM after forced degradation UPLC–MS/MSⁿ, Orbitrap has been used. It was found that new impurities are oxidation products: (S)-1-((S)-2-((S)-1-ethoxy-4-(o,m,p-hydroxyphenyl)-1-oxobutan-2-ylamino)propanoyl)pyrrolidine-2-carboxylic acid, (2S)-1-((2S)-2-((2S)-1-ethoxy-4-hydroxy-1-oxo-4-phenylbutan-2-ylamino)propanoyl)pyrrolidine-2-carboxylic acid. (S)-2-(3-phenylpropylamino)-1-(pyrrolidin-1-yl)propan-1-one was identified as a new degradation impurity.     

Development and validation of a rapid RP-HPLC method for the determination of cetirizine or fexofenadine with pseudoephedrine in binary pharmaceutical dosage forms

The objective of the current study was to develop a simple, accurate, precise and rapid reversed-phase HPLC method and subsequent validation using ICH suggested approach for the determination of antihistaminic-decongestant pharmaceutical dosage forms containing binary mixtures of pseudoephedrine hydrochloride (PSE) with fexofenadine hydrochloride (FEX) or cetirizine dihydrochloride (CET). The chromatographic separation of PSE, FEX and CET was achieved on a Zorbax C8 (150 mm × 4.6 mm; 5 μm particle size) column using UV detection at 218 and 222 nm. The optimized mobile phase was consisted of TEA solution (0.5%, pH 4.5)–methanol–acetonitrile (50:20:30, v/v/v). The retention times were 1.099, 2.714 and 3.808 min for PSE, FEX and CET, respectively. The proposed method provided linear responses within the concentration ranges 30–240 and 1.25–10 μg ml⁻¹ with LOD values of 1.75 and 0.10 μg ml⁻¹ for PSE and CET, respectively. Linearity range for PSE–FEX binary mixtures were 10–80 and 5–40 μg ml⁻¹ with LOD values of 0.75 and 0.27 μg ml⁻¹ for PSE and FEX, respectively. Correlation coefficients (r) of the regression equations were greater than 0.999 in all cases. The precision of the method was demonstrated using intra- and inter-day assay R.S.D. values which were less than 1% in all instances. No interference from any components of pharmaceutical dosage forms or degradation products was observed. According to the validation results, the proposed method was found to be specific, accurate, precise and could be applied to the quantitative analysis of these drugs in capsules containing PSE–CET or extended-release tablets containing PSE–FEX binary mixtures.     

Risk assessment of chlorpyrifos on rice and cabbage in China

Chlorpyrifos is a widely used organophosphorus insecticide in agricultural pest control. To understand the residue behavior of chlorpyrifos and to evaluate the dietary risk of chlorpyrifos residue in food in China, a number of residue studies were conducted on rice and cabbage. The supervised trial median residues (STMRs) for rice and cabbage were less than 0.010 and 0.227 mg kg⁻¹, respectively. Only 7.4% and 13.3% of acceptable daily intake (ADI) (0–0.01 mg kg⁻¹ bw) of chlorpyrifos is occupied by dietary daily intake to the Chinese adult and children, respectively, due to the consumption of rice and cabbage. These results on risk assessment were consistent with that of JMPR. Incorporation of market survey residue data gave a 5-fold reduction in the estimated exposures to chlorpyrifos. Concerning the acute exposure, the national estimated short-term intake (NESTI) represents 0.077% and 10.6% for rice and cabbage, respectively, of the acute reference dose (ARfD) (0–0.1 mg kg⁻¹ bw). The application of chlorpyrifos at the recommended dose on rice and cabbage is unlikely to pose any public health issues if it is applied according to the good agricultural practices (GAPs) established by each country.     

Role of iodine in the toxicity of diiodomethyl-p-tolylsulfone (DIMPTS) in rats: ADME

Metabolism of diiodomethyl-p-tolylsulfone (DIMPTS) was investigated in rats to determine the role of iodide in its toxicity. Fischer 344 (F-344) (5 or 50 mg/kg) or Sprague Dawley (SD) (5 mg/kg) rats were gavaged with ¹⁴C-DIMPTS or dermally applied with 5 mg/kg (F-344 only) and absorption, distribution, metabolism and excretion (ADME) determined. Additional experiments were conducted with its deiodinated analog (methyl-p-tolylsulfone, MPTS) in female F-344 rats (20 mg/kg) for comparison. Orally administered ¹⁴C-DIMPTS was rapidly absorbed and eliminated in urine (92%). The elimination t_½ was 1–4 h. Dermally applied ¹⁴C-DIMPTS remained undetectable in plasma with bioavailability ∼7%, only 5–7% of the dose was recovered in urine. DIMPTS liberated one or both of its iodine atoms upon absorption. The rate of elimination of the liberated iodide from the systemic circulation was 2- to 3-fold slower in SD than F-344 rats, which resulted in higher bioavailability of iodide to SD rats. DIMPTS was primarily oxidized at the benzylic methyl moiety forming the corresponding benzoic acid. Glutathione conjugation on the sulfonyl methyl group, via displacement of I⁻ was also observed. Overall 67–80% of the total iodine atoms were metabolically released from DIMPTS. The MPTS was rapidly absorbed from the GI tract, metabolized and eliminated in urine similar to that of DIMPTS. These data were compared to iodide toxicokinetic results of a reproductive toxicity study for DIMPTS (80 mg/kg/day) and MPTS (32 mg/kg/day), where DIMPTS was toxic to dams and pups, while MPTS caused no toxicity. These data show that the liberated iodide is the ultimate toxicant of DIMPTS, which is readily transported to pups through milk, while the methyltolylsulfone backbone structure (MPTS) of DIMPTS is relatively nontoxic.     

Profiling and quantification of isoflavone-C-glycosides impurities in puerarin injection by liquid chromatography coupled to ESI-ion trap mass spectrometry

An HPLC/DAD/MSⁿ method was established for the qualitative and quantitative analysis of the impurities in puerarin injection (PI), a widely used drug in China. The analytical HPLC was performed on an Agela RP-C18 column using 0.1% aqueous formic acid (v:v) and methanol as mobile phase. A total of nine impurities were detected and eight of them were identified as isoflavone-C-glycosides basing on their UV spectra and MSⁿ spectra and comparing with the literature data. An HPLC method for the assay of two common impurities in the commercial PI samples, i.e., neopuerarin A and neopuerarin B, was then established. The validation of the method, including sensitivity, linearity, precision, accuracy, was carried out. The calibration curves showed good linearity of R² > 0.9999 and LOQ (S/N = 10) were less than 3.73 ng. The precision was evaluated by intra- and inter-day assays and R.S.D. values were less than 0.94%. The average recovery rates were 97.0% and 99.5%, respectively, with R.S.D. less than 1.38%. The contents of neopuerarin A and neopuerarin B in various commercial brands of PI samples varied over the range of 0.30–1.16% and 0.42–1.66%, respectively. This is the first report on the impurities in PI.     

Simultaneous quantification of dextromethorphan and its metabolites dextrorphan, 3-methoxymorphinan and 3-hydroxymorphinan in human plasma by ultra performance liquid chromatography/tandem triple-quadrupole mass spectrometry

A rapid and sensitive ultra performance liquid chromatography/tandem mass spectrometry (UPLC–MS/MS) method has been developed and validated for the simultaneous quantitative determination of dextromethorphan (DM) and its metabolites dextrorphan (DX), 3-methoxymorphinan (3MM) and 3-hydroxymorphinan (3HM), in human lithium heparinized plasma. The extraction involved a simple liquid–liquid extraction with 1 ml n-butylchloride from 200 μl aliquots of plasma, after the addition of 20 μl 4% (v/v) ammonium hydroxide and 100 μl stable labeled isotopic internal standards in acetonitrile. Chromatographic separations were achieved on an Aquity UPLC^® BEH C₁₈ 1.7 μm 2.1 mm × 100 mm column eluted at a flow-rate of 0.250 ml/min on a gradient of acetonitrile. The overall cycle time of the method was 7 min, with elution times of 1.3 min for DX and 3HM, 2.8 min for 3MM and 2.9 min for DM. The multiple reaction monitoring transitions were set at 272 > 215 (m/z), at 258 > 133 (m/z), at 258 > 213 (m/z) and at 244 > 157 (m/z) for DM, DX, 3MM and 3HM, respectively. The calibration curves were linear (r² ≥ 0.995) over the range of 0.500–100 nM with the lower limit of quantitation validated at 0.500 nM for all compounds, which is equivalent to 136, 129, 129 and 122 pg/ml for DM, DX, 3MM and 3HM, respectively. Extraction recoveries were constant, but ranged from 39% for DM to 83% for DX. The within-run and between-run precisions were within 11.6%, while the accuracy ranged from 92.7 to 110.6%. The applicability of the bioanalytical method was demonstrated and is currently implemented in a clinical trial to study DM as probe-drug for individualized tamoxifen treatment in breast cancer patients.