Consumption of the Sugar Substitute Stevia Leads to Cross-Reactivity of CEDIA® Buprenorphine II Immunoassay.

Buprenorphine is a semisynthetic opioid that is often used in opiate maintenance therapy. For this purpose, regular toxicological analyses of urine samples are mandatory. For fast analytical results, analyses are commonly performed by immunoassay, for example, Thermo Scientific™ CEDIA® Buprenorphine or Buprenorphine II assay. One drawback of immunoassay-based methods is the possible cross-reaction with other substances. Several drugs have already been checked for cross-reactivity to CEDIA® Buprenorphine II immunoassay. In contrast, cross-reactivities have not been checked for any food additives. In the present study, a cross-reaction of CEDIA® Buprenorphine II assay to steviol glucuronide was investigated. Steviol glucuronide is a phase II metabolite of the sugar substitute stevia. For our study, 32 urine samples of patients in rehabilitation centers were collected. These samples were tested positive with the CEDIA® Buprenorphine II immunoassay. These findings were suspicious, because it was highly unlikely that the patients in those institutions had access to buprenorphine. The absence or presence of buprenorphine in urine samples was evaluated by a validated gas chromatography-mass spectrometry method. In order to determine the concentration of steviol glucuronide in urine samples, a liquid chromatography-tandem mass spectrometry method has been developed and fully validated according to the respective guidelines of the German Society of Toxicological and Forensic Chemistry. The cross-reactivity of steviol glucuronide in the CEDIA® Buprenorphine II immunoassay was observed at concentrations above 15,000 µg/L. These findings demonstrate that food additives should also be considered as compounds that may reduce the selectivity of immunoassays and emphasize the importance of confirming implausible results by selective analytical methods.

A Novel Enzyme Immunoassay for the Detection of Buprenorphine, Norbuprenorphine and Their Glucuronides in Urine.

Buprenorphine is a commonly used opioid in pain therapy as well as in opiate maintenance therapy. Immunoassays are quick and cost-effective methods for the necessary toxicological urine analysis of maintenance therapy patients. In this study a novel enzymatic immunoassay, the Thermo Fisher Scientific CEDIA Buprenorphine II assay (Bup2) was evaluated for the detection of buprenorphine, norbuprenorphine and their conjugated metabolites in human urine samples. The Bup2 assay has a cut-off of 10 ng/mL with ±25% controls, whereas the existing CEDIA Buprenorphine assay (Bup1) has a cut-off of 5 ng/mL and ±40% controls. Both assays were analyzed on a Thermo Scientific Indiko Plus benchtop analyzer. Seven-day precision studies of Bup2 assay demonstrated excellent precision of 7.2-10.6%. No crossover between control samples and the cut-off level were observed. Urine samples of 120 patients undergoing opiate maintenance therapy were collected. Immunoassay results of Bup1 and Bup2 were confirmed by gas chromatography mass spectrometry (GC/MS) for buprenorphine and norbuprenorphine as well as for their glucuronides. Comparison showed a specificity of 0.99 between the Bup2 assay and GC/MS, whereas the Bup1 assay had a specificity 0.70 due to 21 false positive samples. The reason is a known cross-reactivity of the Bup1 assay to opiate compounds. The Bup2 assay revealed one false positive result close to the cut-off value; no specific candidate possibly causing a cross-reaction was detected by GC/MS and liquid chromatography tandem mass-spectrometry (LC/MS/MS) methods. The data presented demonstrate an excellent correlation of the Bup2 assay to GC/MS, showing improved specificity and sensitivity when compared to the Bup1 assay. Thus, the Bup2 assay is highly suitable for urine testing, even for opiate maintenance patients receiving high doses of morphine.

Does obesity interfere with anastrozole treatment? Positive association between body mass index and anastrozole plasma levels.

INTRODUCTION: The efficacy of adjuvant endocrine treatment with aromatase inhibitors (AIs), inhibiting the conversion of androgens to estrogen in adipose tissue, might depend on the overall volume of adipose tissue. However, little evidence is available regarding the pharmacokinetic behavior of AIs in women with obesity. The aim of this study was to investigate the interaction between body mass index (BMI) and anastrozole treatment as well as estrogenic activity. PATIENTS AND METHODS: A total of 216 postmenopausal patients with early-stage breast cancer who were receiving AI treatment with anastrozole constituted the final sample included in the analysis. During a regular 3-month after-care check-up, sociodemographic and clinical data and BMI were assessed. Blood samples were collected during routine blood testing. Measurement of AI plasma levels was performed by liquid chromatography-tandem mass spectrometry. Follicle stimulating hormone (FSH) and estradiol were measured within the routine blood examination. RESULTS: A median anastrozole plasma concentration of 34.7 ng/mL (mean, 37.4), with a large interindividual variability, was observed (SD, 15.1; range, 5.4-86.5). After age adjustment, it was found that anastrozole plasma concentrations significantly increased with BMI (r = 0.241; P = .001). Anastrozole serum concentrations in women with obesity (BMI ≥ 30) exceeded those of women with normal weight (BMI ≤ 25) by 25%. Women with excess weight had lower mean FSH levels, indicating higher estrogenic activity, compared with women with normal weight. CONCLUSION: This study indicates that BMI is a vital factor in anastrozole metabolism, as measured by anastrozole plasma concentration and FSH levels. Further research is mandatory to clarify results on the association of obesity and AI treatment efficacy to allow adapting AI treatment accordingly.

Detection and identification of drugs and toxicants in human body fluids by liquid chromatography-tandem mass spectrometry under data-dependent acquisition control and automated database search.

Systematic toxicological analysis (STA) is aimed at detecting and identifying all substances of toxicological relevance (i.e. drugs, drugs of abuse, poisons and/or their metabolites) in biological material. Particularly, gas chromatography-mass spectrometry (GC/MS) represents a competent and commonly applied screening and confirmation tool. Herein, we present an untargeted liquid chromatography-tandem mass spectrometry (LC/MS/MS) assay aimed to complement existing GC/MS screening for the detection and identification of drugs in blood, plasma and urine samples. Solid-phase extraction was accomplished on mixed-mode cartridges. LC was based on gradient elution in a miniaturized C18 column. High resolution electrospray ionization-MS/MS in positive ion mode with data-dependent acquisition control was used to generate tandem mass spectral information that enabled compound identification via automated library search in the "Wiley Registry of Tandem Mass Spectral Data, MSforID". Fitness of the developed LC/MS/MS method for application in STA in terms of selectivity, detection capability and reliability of identification (sensitivity/specificity) was demonstrated with blank samples, certified reference materials, proficiency test samples, and authentic casework samples.

Adherence evaluation of endocrine treatment in breast cancer: methodological aspects.

BACKGROUND: Current studies on adherence to endocrine therapy in breast cancer patients suffer from methodological limitations due to a lack of well-validated methods for assessing adherence. There is no gold standard for measuring adherence. The aim of our study was to compare four different approaches for evaluating adherence to anastrozole therapy for breast cancer with regard to concordance between methods. METHODS: Outpatients with early breast cancer treated with anastrozole completed the multi-method assessment of adherence. We implemented a self-report scale (the Simplified Medication Adherence Questionnaire), physician- ratings, refill records and determination of anastrozole serum concentration. RESULTS: Comparison of the four approaches using Spearman rank correlation revealed poor concordance across all methods reflecting weak correlations of 0.2-0.4. Considering this data incomparability across methods, we still observed high adherence rates of 78%-98% across measures. CONCLUSION: Our findings contribute to the growing body of knowledge on the impact that methodological aspects exert on the results of adherence measurement in breast cancer patients receiving endocrine treatment. Our findings suggest that the development and validation of instruments specific to patients receiving endocrine agents is imperative in order to arrive at a more accurate assessment and to subsequently obtain more precise estimates of adherence rates in this patient population.

Impact of solvent conditions on separation and detection of basic drugs by micro liquid chromatography-mass spectrometry under overloading conditions.

In this study the impact of solvent conditions on the performance of µLC/MS for the analysis of basic drugs was investigated. Our aim was to find experimental conditions that enable high-performance chromatographic separation particularly at overloading conditions paired with a minimal loss of mass spectrometric detection sensitivity. A focus was put on the evaluation of the usability of different kinds of acidic modifiers (acetic acid (HOAc), formic acid (FA), methansulfonic acid (CH3SO3H), trifluoroacetic acid (TFA), pentafluoropropionic acid (PFPA), and heptafluorobutyric acid (HFBA)). The test mixture consisted of eleven compounds (bunitrolol, caffeine, cocaine, codeine, diazepam, doxepin, haloperidol, 3,4-methylendioxyamphetamine, morphine, nicotine, and zolpidem). Best chromatographic performance was obtained with the perfluorinated acids. Particularly, 0.010-0.050% HFBA (v/v) was found to represent a good compromise in terms of chromatographic performance and mass spectrometric detection sensitivity. Compared to HOAc, on average a 50% reduction of the peak widths was observed. The use of HFBA was particularly advantageous for polar compounds such as nicotine; only with such a hydrophobic ion-pairing reagent chromatographic retention of nicotine was observed. Best mass spectrometric performance was obtained with HOAc and FA. Loss of detection sensitivity induced by HFBA, however, was moderate and ranged from 0 to 40%, which clearly demonstrates that improved chromatographic performance is able to compensate to a large extent the negative effect of reduced ionization efficiency on detection sensitivity. Applications of µLC/MS for the qualitative and quantitative analysis of clinical and forensic toxicological samples are presented.

Development and validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of tamoxifen, anastrozole, and letrozole in human plasma and its application to a clinical study.

There is substantial evidence that circulating estrogens promote the proliferation of breast cancer. Consequently, adjuvant hormonal treatment strategies targeting estrogen action have been established. Such hormonal therapies include selective estrogen receptor modulators, such as tamoxifen, which interfere at the estrogen receptors directly, or non-steroidal aromatase inhibitors, such as anastrozole and letrozole, which inhibit estrogen synthesis through blocking the aromatase, a key enzyme of estrogen production. Despite considerable therapeutic success, in several cases, the use of these drugs is limited by side effects that have been described to significantly impair the adherence of patients to endocrine treatment. However, objective data concerning patient adherence and its clinical relevance are limited. One promising approach to check patient-reported adherence is drug monitoring in human plasma. Therefore, a liquid chromatography-tandem mass spectrometry method to determine the plasma concentrations of tamoxifen, anastrozole, and letrozole has been developed and fully validated according to guidelines for clinical and forensic toxicology. The validation criteria evaluated were selectivity, linearity, accuracy and precision, limit of quantification, recovery and matrix effects, sample stability, and carryover. The six-point calibration curves showed linearity over the range of concentrations from 25 to 500 ng/ml for tamoxifen, 5 to 200 ng/ml for anastrozole, and 10 to 300 ng/ml for letrozole. The intra- and inter-day precision and accuracies were always better than 15%. The validated procedure was successfully applied to a clinical study (Patient-Reported Outcomes in Breast Cancer Patients undergoing Endocrine Therapy, PRO-BETh). A major aim of PRO-BETh study is the comprehensive evaluation of adherence to treatment in pre- and post-menopausal women with breast cancer. Plasma samples of 310 breast cancer patients undergoing anti-estrogen therapy were analyzed. Eight samples did not contain a quantifiable amount of drug, strongly indicating non-adherence of the corresponding patients to adjuvant breast cancer treatment. Furthermore, plasma concentrations at the lower end of the observed plasma level distribution might represent a hint but not a confirmation for non-adherence in terms of non-daily and irregular intake of the prescribed drug.

Comprehensive identification of active compounds in tablets by flow-injection data-dependent tandem mass spectrometry combined with library search.

A convenient mass spectrometric approach for the identification of toxicologically relevant compounds in tablets and tablet residues is presented. For comprehensive forensic-toxicological analysis electrospray ionization mass spectrometry was accomplished in positive as well as in negative ion mode on a quadrupole-quadrupole-time-of-flight instrument. Dissolved samples were introduced into the mass spectrometer by flow-injection. Mass spectra as well as tandem mass spectra were acquired. A data-dependent acquisition strategy was used to switch between the mass spectrometric modes. Identification was accomplished via search within a tandem mass spectral library. The applied database contained 8252 spectra collected from 836 compounds in positive ion mode as well as 1023 spectra collected from 103 compounds in negative ion mode. A total of 22 casework samples collected during autopsies from mouth, oesophagus or gastric contents, seized by the police, or found with patients at hospital were screened. Twelve samples contained compounds only detectable in positive ion mode (sildenafil, dihydrocodeine, diphenhydramine, oxprenolol, N-methyl-3,4-methylenedioxyamphetamine, morphine, amphetamine, caffeine, pemoline, orphenadrine, m-chlorphenylpiperazine and tramadol), six samples contained species exclusively detectable in negative ion mode (salicylic acid, acetylsalicylic acid, ibuprofen, ketorolac, valproic acid and phenobarbital), and three samples contained diclofenac detectable in both ionization polarities. One sample did not contain any compound amenable to mass spectrometric analysis. For verification all samples were additionally analyzed by GC/MS. Both methods revealed identical results for all but one sample. The beta-adrenergic blocker oxprenolol was exclusively detected by the flow-injection method.

Metabolic studies of tetrazepam based on electrochemical simulation in comparison to in vivo and in vitro methods.

During the last 2 years, the knowledge on the metabolic pathway of tetrazepam, a muscle relaxant drug, was expanded by the fact that diazepam was identified as a degradation product of tetrazepam. The present study demonstrates that this metabolic conversion, recently discovered by in vivo studies, can also be predicted on the basis of a purely instrumental method, consisting of an electrochemical cell (EC) coupled to online liquid chromatography (LC) and mass spectrometry (MS). By implementing a new electrochemical cell type into the EC-LC-MS set-up and by an enhanced oxidation potential range up to 2V, one limitation of the electrochemical metabolism simulation, the hydroxylation of alkanes and alkenes, has been overcome. Instead of commonly used flow-through cell with a porous glassy carbon working electrode, a wall-jet cell with exchangeable electrode material was used for this study. Thereby, the entire metabolic pathway of tetrazepam, in particular including the hydroxylation of the tetrazepam cyclohexenyl moiety, was simulated. The electrochemical results were not only compared to microsomal incubations, but also to in vivo experiments, by analysing urine samples from a patient after tetrazepam delivery. For structure elucidation of the detected metabolites, MS/MS experiments were performed. The comparison of electrochemistry to in vitro as well as to in vivo experiments underlines the high potential of electrochemistry as a fast screening tool in the prediction of metabolic transformations in drug development.

On the inter-instrument and the inter-laboratory transferability of a tandem mass spectral reference library: 2. Optimization and characterization of the search algorithm.

A sophisticated matching algorithm developed for highly efficient identity search within tandem mass spectral libraries is presented. For the optimization of the search procedure a collection of 410 tandem mass spectra corresponding to 22 compounds was used. The spectra were acquired in three different laboratories on four different instruments. The following types of tandem mass spectrometric instruments were used: quadrupole-quadrupole-time-of-flight (QqTOF), quadrupole-quadrupole-linear ion trap (QqLIT), quadrupole-quadrupole-quadrupole (QqQ), and linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer (LIT-FTICR). The obtained spectra were matched to an established MS/MS-spectral library that contained 3759 MS/MS-spectra corresponding to 402 different reference compounds. All 22 test compounds were part of the library. A dynamic intensity cut-off, the search for neutral losses, and optimization of the formula used to calculate the match probability were shown to significantly enhance the performance of the presented library search approach. With the aid of these features the average number of correct assignments was increased to 98%. For statistical evaluation of the match reliability the set of fragment ion spectra was extended with 300 spectra corresponding to 100 compounds not included in the reference library. Performance was checked with the aid of receiver operating characteristic (ROC) curves. Using the magnitude of the match probability as well as the precursor ion mass as benchmarks to rate the obtained top hit, overall correct classification of a compound being included or not included in the mass spectrometric library, was obtained in more than 95% of cases clearly indicating a high predictive accuracy of the established matching procedure.

On the inter-instrument and inter-laboratory transferability of a tandem mass spectral reference library: 1. Results of an Austrian multicenter study.

The inter-instrument and inter-laboratory transferability of a tandem mass spectral reference library originally built on a quadrupole-quadrupole-time-of-flight instrument was examined. The library consisted of 3759 MS/MS spectra collected from 402 reference compounds applying several different collision-energy values for fragmentation. In the course of the multicenter study, 22 test compounds were sent to three different laboratories, where 418 tandem mass spectra were acquired using four different instruments from two manufacturers. The study covered the following types of tandem mass spectrometers: quadrupole-quadrupole-time-of-flight, quadrupole-quadrupole-linear ion trap, quadrupole-quadrupole-quadrupole, and linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer. In each participating laboratory, optimized instrumental parameters were gathered solely from routinely applied workflows. No standardization procedure was applied to increase the inter-instrument comparability of MS/MS spectra. The acquired tandem mass spectra were matched against the established reference library using a sophisticated matching algorithm, which is presented in detail in a companion paper. Correct answers, meaning that the correct compound was retrieved as top hit, were obtained in 98.1% of cases. For the remaining 1.9% of spectra, the correct compound was matched at second rank. The observed high percentage of correct assignments clearly suggests that the developed mass spectral library search approach is to a large extent platform independent.

Unraveling the metabolic transformation of tetrazepam to diazepam with mass spectrometric methods.

The metabolic transformation pathways of the 1,4-benzodiazepine tetrazepam (C(16)H(17)ClN(2)O, average mass: 288.772) were studied with capillary LC-QqTOF-MS and -MS/MS by analyzing human plasma and urine samples collected from healthy volunteers. Each volunteer took 50 mg of tetrazepam, given in the form of one tablet of Myolastan (Sanofi-Synthelabo, Vienna, Austria). Accurate molecular mass measurements in full-scan mode (scan range: 50-700) were used to survey the collected samples for putative metabolic transformation products. Full-scan fragment ion mass spectra were collected in subsequent LC/MS/MS experiments. Each spectrum was matched to a spectral library containing 3759 MS/MS-spectra of 402 compounds, including eighteen different benzodiazepines, to prove the structural relatedness of a tentative metabolite to tetrazepam. This "similarity search" approach provided a rapid and powerful tool to exclude non-drug-related species, even without any knowledge of the fragmentation chemistry. Interpretation of tandem mass spectrometric data was only required in order to elucidate the site of transformation. Using this strategy, 11 major classes of tetrazepam metabolites were identified. Possible metabolic routes from tetrazepam to diazepam (C(16)H(13)ClN(2)O, average mass: 284.740) via repeated hydroxylation and dehydration of the cylohexenyl moiety were discovered. No evidence for extensive hydroxylation of tetrazepam at position 3 of the diazepine ring was found. In contrast to what is commonly believed, this distinct transformation reaction may be of only minor importance. Furthermore, the occurrence of demethylation, hydration, and glucuronidation reactions was proven.

Polyacetylenes from the Apiaceae vegetables carrot, celery, fennel, parsley, and parsnip and their cytotoxic activities.

A dichloromethane extract of root celery yielded falcarinol, falcarindiol, panaxydiol, and the new polyacetylene 8-O-methylfalcarindiol. The structure of the new compound was established by one- and two-dimensional (1D and 2D) NMR, mass spectrometry, and optical rotation data. Nonpolar extracts of roots and bulbs of carrots, celery, fennel, parsley, and parsnip were investigated for their content of polyacetylenes by high-performance liquid chromatography with diode array detection (HPLC-DAD). All five species contained polyacetylenes, although carrots and fennel only in minor amounts. Additionally, the cytotoxicity of the four polyacetylenes against five different cell lines was evaluated by the annexin V-PI assay. Falcarinol proved to be the most active compound with a pronounced toxicity against acute lymphoblastic leukemia cell line CEM-C7H2, with an IC(50) of 3.5 micromol/L. The possible chemopreventive impact of the presented findings is discussed briefly.