Towards clinical adherence monitoring of oral endocrine breast cancer therapies by LC-HRMS-method development, validation, comparison of four sample matrices, and proof of concept.

Oral endocrine therapies (OET) for breast cancer treatment need to be taken over a long period of time and are associated with considerable side effects. Therefore, adherence to OET is an important issue and of high clinical significance for breast cancer patients' caregivers. We hypothesized that a new bioanalytical strategy based on liquid chromatography and high-resolution mass spectrometry might be suitable for unbiased adherence monitoring (AM) of OET. Four different biomatrices (plasma, urine, finger prick blood by volumetric absorptive microsampling (VAMS), oral fluid (OF)) were evaluated regarding their suitability for AM of the OET abemaciclib, anastrozole, exemestane, letrozole, palbociclib, ribociclib, tamoxifen, and endoxifen. An analytical method was developed and validated according to international recommendations. The analytical procedures were successfully validated in all sample matrices for most analytes, even meeting requirements for therapeutic drug monitoring. Chromatographic separation of analytes was achieved in less than 10 min and limits of quantification ranged from 1 to 1000 ng/mL. The analysis of 25 matching patient samples showed that AM of OET is possible using all four matrices with the exception of, e.g., letrozole and exemestane in OF. We were able to show that unbiased bioanalytical AM of OET was possible using different biomatrices with distinct restrictions. Sample collection of VAMS was difficult in most cases due to circulatory restraints and peripheral neuropathy in fingers and OF sampling was hampered by dry mouth syndrome in some cases. Although parent compounds could be detected in most of the urine samples, metabolites should be included when analyzing urine or OF. Plasma is currently the most suitable matrix due to available reference concentrations.

Undeclared Doping Substances are Highly Prevalent in Commercial Sports Nutrition Supplements.

Sports nutrition supplements have previously been reported to contain undeclared doping substances. The use of such supplements can lead to general health risks and may give rise to unintentional doping violations in elite sports. To assess the prevalence of doping substances in a range of high-risk sports nutrition supplements available from Dutch web shops. A total of 66 sports nutrition supplements - identified as potentially high-risk products claiming to modulate hormone regulation, stimulate muscle mass gain, increase fat loss, and/or boost energy - were selected from 21 different brands and purchased from 17 web shops. All products were analyzed for doping substances by the UK life sciences testing company LGC, formerly known as the Laboratory of the Government Chemist, using an extended version of their ISO17025 accredited nutritional supplement screen. A total of 25 out of the 66 products (38%) contained undeclared doping substances, which included high levels of the stimulants oxilofrine, ß-methylphenethylamine (BMPEA) and N,ß-dimethylphenethylamine (NBDMPEA), the stimulant 4-methylhexan-2-amine (methylhexaneamine, 1,3-dimethylamylamine, DMAA), the anabolic steroids boldione (1,4-androstadiene-3,17-dione) and 5-androstene-3ß,17α-diol (17α-AED), the beta-2 agonist higenamine and the beta-blocker bisoprolol. Based upon the recommended dose and the potential variability of analyte concentration, the ingestion of some products identified within this study could pose a significant risk of unintentional doping violations. In addition to inadvertent doping risks, the prescribed use of 3 products (4.5%) could likely impose general health risks.

Genetic variation in a human odorant receptor alters odour perception.

Human olfactory perception differs enormously between individuals, with large reported perceptual variations in the intensity and pleasantness of a given odour. For instance, androstenone (5alpha-androst-16-en-3-one), an odorous steroid derived from testosterone, is variously perceived by different individuals as offensive ("sweaty, urinous"), pleasant ("sweet, floral") or odourless. Similar variation in odour perception has been observed for several other odours. The mechanistic basis of variation in odour perception between individuals is unknown. We investigated whether genetic variation in human odorant receptor genes accounts in part for variation in odour perception between individuals. Here we show that a human odorant receptor, OR7D4, is selectively activated in vitro by androstenone and the related odorous steroid androstadienone (androsta-4,16-dien-3-one) and does not respond to a panel of 64 other odours and two solvents. A common variant of this receptor (OR7D4 WM) contains two non-synonymous single nucleotide polymorphisms (SNPs), resulting in two amino acid substitutions (R88W, T133M; hence 'RT') that severely impair function in vitro. Human subjects with RT/WM or WM/WM genotypes as a group were less sensitive to androstenone and androstadienone and found both odours less unpleasant than the RT/RT group. Genotypic variation in OR7D4 accounts for a significant proportion of the valence (pleasantness or unpleasantness) and intensity variance in perception of these steroidal odours. Our results demonstrate the first link between the function of a human odorant receptor in vitro and odour perception.

On-chip microelectrophoresis for the study of in vitro nonhomologous end-joining DNA double-strand break repair.

Oligomerization of linearized plasmids by nuclear proteins extracts, a recognized measure of nonhomologous end-joining (NHEJ) repair capacity, is typically assessed through agarose gel electrophoresis, a labor-intensive procedure. In the current study, a more convenient NHEJ assay was developed using microchips that allow scaled-down separation and quantification. This microchip method allows a considerable reduction in sample amount and analysis time with similar costs and comparable or slightly better precision. Data obtained with quercetin and wortmannin show that the method can be applied to the screening of food components and natural products for positive and negative modulators of NHEJ, potential chemopreventive and indirect genotoxic compounds, respectively.

Analysis of fluticasone propionate in induced sputum by mass spectrometry.

Although evaluation of induced sputum has shown promise as a marker of eosinophilic airway inflammation in asthmatic subjects, most studies, to date, do not adequately address the potential effect that inhaled corticosteroids may have on sputum eosinophilia. This study was designed to prospectively evaluate analysis of fluticasone propionate (FP) in whole sputum by mass spectrometry as a tool to determine recent administration of inhaled FP. Induced sputum of nonsmoking asthmatic subjects was prospectively analyzed 16-24 hours after witnessed administration of orally inhaled FP. FP was extracted from whole sputum via an acetonitrile protein precipitation followed by methylene chloride liquid extraction of the supernatant (AB 4000; AB Sciex). A portion of the reconstituted sample was analyzed by liquid chromatography tandem mass spectrometry using a triple quad tandem mass spectrometer. Results were compared with those from nonsmoking asthmatic subjects not receiving inhaled FP. Twenty-two asthmatic subjects on FP and 9 asthmatic subjects without FP underwent sputum induction 16-24 hours following witnessed administration of FP. Sufficient sputum for analysis was obtained from 30 of 31 subjects. FP was detected in 22 of 22 asthmatic subjects receiving FP (range, 29-133,000 pg/mL) and was undetectable in 8 of 8 subjects not receiving FP. The sensitivity and specificity of tandem mass spectrometry's ability to detect FP in sputum was 100% and 100%, respectively. Analysis of FP in induced sputum is a reliable method to verify recent administration of inhaled FP. Induced asthmatic sputum from one induction may be used to concomitantly assess sputum eosinophilia as well as recent administration of FP.

A high-throughput liquid chromatography tandem mass spectrometry method for the comparative determination of fluticasone propionate by reversed-phase liquid chromatography and capillary electrophoresis methods in pharmaceutical nasal sprays.

A simple, specific, rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of fluticasone propionate (FP) in pharmaceutical formulations was developed and validated using deflazacort as internal standard (IS). The LC-MS/MS method was carried out on a C8 column (50 mm) with a mobile phase consisted of methanol : water (95 : 5, v/v) 100 mM formic acid-50 mM ammonium acetate (90 : 5 : 5, v/v/v). The mass spectrometry method was performed employing positive atmospheric pressure chemical ionization technique, operating in multiple reaction monitoring mode. The chromatographic separation was obtained within 1.5 min and it was linear in the concentration range of 10-1000 ng mL(-1). Moreover, method validation demonstrates acceptable results for the specificity, accuracy, precision and robustness. The proposed method was successfully applied for the quantitative analysis of FP nasal sprays and the results were compared to validated liquid chromatography and capillary electrophoresis methods with photodiode array detectors showing non-significant difference (P > 0.05).

Identification of the designer steroid Androsta-3,5-diene-7,17-dione in a dietary supplement.

A liquid chromatography-mass spectrometry (LC-MS) screen for known anabolic-androgenic steroids in a dietary supplement product marketed for "performance enhancement" detected an unknown compound having steroid-like spectral characteristics. The compound was isolated using high performance liquid chromatography with ultraviolet detection (HPLC-UV) coupled with an analytical scale fraction collector. After the compound was isolated, it was then characterized using gas chromatography with simultaneous Fourier Transform infrared detection and mass spectrometry (GC-FT-IR-MS), liquid chromatography-high resolution accurate mass-mass spectrometry (LC-HRAM-MS) and nuclear magnetic resonance (NMR). The steroid had an accurate mass of m/z 285.1847 (error-0.57 ppm) for the protonated species [M + H]+ , corresponding to a molecular formula of C19 H24 O2 . Based on the GC-FT-IR-MS data, NMR data, and accurate mass, the compound was identified as androsta-3,5-diene-7,17-dione. Although this is not the first reported identification of this designer steroid in a dietary supplement, the data provided adds information for identification of this compound not previously reported. This compound was subsequently detected in another dietary supplement product, which contained three additional active ingredients.

Tyrosine Induced Metabolome Alterations of Penicillium roqueforti and Quantitation of Secondary Key Metabolites in Blue-Mold Cheese.

To map qualitative and quantitative metabolome alterations when Penicillium roqueforti is grown in an environment where l-tyrosine levels are perturbed, the recently established differential off-line LC-NMR (DOLC-NMR) approach was successfully applied in connection with an absolute metabolite quantitation using a quantitative 1H NMR protocol following the ERETIC 2 (Electronic REference To access In vivo Concentrations) methodology. Among the 23 influenced metabolites, amino acid degradation products like 2-(4-hydroxyphenyl)acetic acid and 2-(3,4-dihydroxyphenyl)acetic acid underwent a tremendous upregulation in the amino acid perturbed approach. Moreover, the output of secondary metabolites like andrastin A, eremofortin B, and the tetrapeptide d-Phe-l-Val-d-Val-l-Tyr was affected in the case of the presence or absence of the added aromatic amino acid. Furthermore, the isolated secondary metabolites of P. roqueforti have been quantified for the first time in five divergent Penicillium isolates by means of a validated LC-ECHO-MS/MS method. This technique is used to compensate the effect of co-extracted matrix compounds during the analysis and to utilize quasi-internal standards to quantify all metabolites of interest accurately. This screening outlined the great variety between the different fungi of the same species. The metabolite spectra of wild-type fungi included more toxic intermediates compared to a selected fungi used as a starter culture for blue-mold cheese production. In addition, these secondary metabolites were quantified in commercially available white- and blue-mold cheese samples. The main differences between the analyte profiles of white and blue cheeses were linked to the impact of the used starter culture. Specific metabolites detected from P. roqueforti like andrastin A and B or roquefortine C could not be detected in white cheese. Among the blue cheese samples, different metabolite pattern could be observed regarding various P. roqueforti starter cultures.

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.

Evidence that androstadienone, a putative human chemosignal, modulates women's attributions of men's attractiveness.

Considerable research effort has focused on whether specific compounds found within human body odor influence the behavior or physiology of other individuals. The most intensively studied is 4,16-androstadien-3-one, a chemical which is known to modulate mood and have activational effects in the sympathetic nervous system in a context-dependent manner, but whose action in mate-choice contexts remains largely untested. Here we present evidence that this androgen steroid may modulate women's judgments of men's attractiveness in an ecologically valid context. We tested the effects of androstadienone at a speed-dating event in which men and women interacted in a series of brief dyadic encounters. Men were rated more attractive when assessed by women who had been exposed to androstadienone, an effect that was seen in two out of three studies. The results suggest that androstadienone can influence women's attraction to men, and also that research into the modulatory effects of androstadienone should be made within ecologically valid contexts.

Development of a rapid normal-phase LC-positive ion APCI-MS method for simultaneous detection and quantitation of cholesterol, androst-4-ene-3,1 7-dione, and androsta-1,4-diene-3,17-dione.

This paper describes the development of a normal-phase liquid chromatograph-UV-diode array detection-positive ion atmospheric pressure chemical ionization-mass spectrometry method for the simultaneous identification and quantitation of cholesterol, androst-4-ene-3,17-dione (AD), and androsta-1,4-diene-3,17-dione (ADD) in fermentation broths. The compounds detected under positive ion atmospheric pressure chemical ionization on a mass spectrometer by selected ion monitoring are separated by normal-phase high-performance liquid chromatography. [M+H]+ ions are taken into consideration for quantitation of AD and ADD, and [M-H2O+H]+ ions are considered for quantitation of cholesterol. The compounds are analyzed on a Si60 silica (5 microm, 125 x 4-mm i.d.) Merck column using a 2:3 isocratic mixture of isopropyl alcohol and hexane. The calibration curves resulting from the reference compounds in the concentration range of 100-5000 pg on column exhibit a good linear correlation (r2 > or = 0.996). The method is validated by analyzing six replicates of broth samples fortified with three compounds, namely, cholesterol, AD, and ADD, at 0.050 and 0.5 microg/g levels. The mean recoveries for the fortifications range from 90% to 98% with relative standard deviations in the range of 3.36% to 9.78%. The method is developed to study the qualitative as well as quantitative conversion of cholesterol to AD and ADD by a microorganism identified as Nocardia sp. These studies helped the investigation of the reaction kinetics, which showed that the molar biotransformation of cholesterol into AD and ADD was 21%, even when the reaction was prolonged for 96 h.

Steroidogenic Metabolism of Galeterone Reveals a Diversity of Biochemical Activities.

Galeterone is a steroidal CYP17A1 inhibitor, androgen receptor (AR) antagonist, and AR degrader, under evaluation in a phase III clinical trial for castration-resistant prostate cancer (CRPC). The A/B steroid ring (Δ5,3ß-hydroxyl) structure of galeterone is identical to that of cholesterol, which makes endogenous steroids with the same structure (e.g., dehydroepiandrosterone and pregnenolone) substrates for the enzyme 3ß-hydroxysteroid dehydrogenase (3ßHSD). We found that galeterone is metabolized by 3ßHSD to Δ4-galeterone (D4G), which is further converted by steroid-5α-reductase (SRD5A) to 3-keto-5α-galeterone (5αG), 3α-OH-5α-galeterone, and 3ß-OH-5α-galeterone; in vivo it is also converted to the three corresponding 5ß-reduced metabolites. D4G inhibits steroidogenesis and suppresses AR protein stability, AR target gene expression, and xenograft growth comparably with galeterone, and further conversion by SRD5A leads to loss of several activities that inhibit the androgen axis that may compromise clinical efficacy. Together, these findings define a critical metabolic class effect of steroidal drugs with a Δ5,3ß-hydroxyl structure.

Validation of a reverse-phase high performance liquid chromatographic method for concurrent assay of a weak base (salmeterol xinafoate) and a pharmacologically active steroid (fluticasone propionate).

The analysis of weakly basic drugs such as salmeterol xinafoate (SX) by reverse-phase liquid chromatography remains a problem, particularly when present in combination with other drugs such as steroids and weak acids. This study describes the validation of an assay for a weakly basic drug, salmeterol (SB), its weakly acidic counter-ion, 1-hydroxy-2-naphthoic acid (XA), and the neutral glucocorticoid, fluticasone propionate (FP) using a second-generation silica stationary phase (Inertsil ODS-2). The assay utilized an Inertsil ODS-2 base-deactivated 250 mm x 4.6mm, 5 microm HPLC column, with 75:25 methanol:0.6% aqueous ammonium acetate as the mobile phase. Under these near neutral conditions, SB demonstrated a good peak shape (tailing factor=1.21+/-0.02, n=85). The method provided a short analysis time: XA, t(R)=2.96 min; SB, t(R)=5.23 min and FP, t(R)=7.01 min. The assay displayed good sensitivity for both XA (LOD for SX=0.22 microgmL(-1)) and SB (LOD for SX=0.26 microgmL(-1)). The limit of detection for FP was 0.19 microgmL(-1). Neither of the drugs was found to interfere in the determination of the other and the assay accuracy (% recovery) was high (the recoveries were: 99.58+/-1.85% for XA, 99.49+/-1.88% for SB and 100.24+/-1.28% for FP). The assay reproducibility was determined with a mean coefficient of variance for the five calibration concentrations of XA=0.71+/-0.18%; SB=1.11+/-0.64% and FP=0.92+/-0.14%. Analysis of a pressurized metered dose inhaler formulation demonstrated recovery of the analytes that are within pharmacopoeial limits. It was shown that RP-HPLC was suitable for the high throughput analysis of the combination of SX and FP.

Determination of the androgenic potency of whole effluents using mosquitofish and trout bioassays.

This study combined bioassay-derived and direct chemical analysis of steroidal compounds in pulp and paper and municipal sewage effluent in order to determine the cause of masculinization of female mosquitofish. The bioassays used in this study consisted of androgen and estrogen receptor binding, and aromatase inhibition using tissues from rainbow trout. This study observed no masculinization of female mosquitofish from a pulp and paper mill effluent that was previously observed to cause this effect. Mosquitofish sampled from the receiving environment of the same mill verified that masculinization was not occurring in the wild. The municipal sewage effluent also had no masculinizing effect. In vitro bioassays indicated significant sources of both androgens and estrogens in the effluents tested with sewage effluent having both the highest estradiol (41 ng/L) and testosterone (182 ng/L) equivalent concentration. These results could not be attributed to any particular compounds measured in the effluents. Two compounds implicated in the masculinization of mosquitofish by pulp and paper effluent, androstenedione and androstadienedione required relatively large (10-100 microg/L) waterborne concentrations to elicit a masculinizing effect and neither of these compounds are likely to occur at levels this high in the natural environment. The potent aromatase inhibitor, 4-hydroxyandrostenedione also did not cause masculinization at 100 microg/L indicating that masculinization did not occur through this mechanism. The mammalian anti-androgen, cyproterone acetate was only partially effective in mosquitofish and reduced the severity of masculinization in the presence of methyl testosterone. While neither effluent masculinized mosquitofish, there was a significant reduction of in vitro ovarian steroid production with the most severe effects observed with the sewage effluent. Overall, this study found the disappearance of a masculinizing effect that had been previously observed; concluded that based on 21 days aqueous exposures androstenedione and androstadienedione are not likely candidates for mosquitofish masculinization, and showed that masculinization and in vitro steroid production are unrelated biological endpoints.

[Analysis of the factors associated with drugs remaining in the Diskhaler following inhalation of fluticasone propionate].

Because it is well known that drug remains in the fluticasone propionate Diskhaler (FP-DH) following a single inhalation, the following patient information is recommended. "Please inhale more than once or twice if any drug remains in the device after inhalation". It is believed the inspiratory flow rate of the individual patient has an influence on the amount of drug that remains in the device. If the dosing performance of FP-DH is dependent on inspiratory effort, establishment of a method of inhalation that makes it independent of inspiratory flow rate is important in clinical practice. In the present study, we investigated the influence of various methods of inhalation of drug remaining in the FP-DH. No significant differences were observed regarding the drug remaining in the device among the inhalation times examined (range, 0.5-2.5 s) or the number of inhalations (range, 1-3 times). On the other hand, the amount of drug remaining in the device did decrease by tapping the device before the second inhalation. The results suggest that the amount of drug remaining in the device can be decreased by tapping the device after the first inhalation if the patient's inspiratory flow rate is low.

HPLC Methods for Quantitation of Exemestane-Luteolin and Exemestane-Resveratrol Mixtures in Nanoformulations.

Two HPLC-DAD assays for the simultaneous quantitation of exemestane (EXE) and resveratrol (RES)-Mix 1-and EXE and luteolin (LUT)-Mix 2-in novel breast cancer therapy nanoformulations were developed. Calibration curves 15-30 µg/mL and samples were injected through an Inertsil ODS-3 (250 × 4.6 mm, 5 µm) column. The gradient elution for Mix 1 was methanol : 0.05% (v/v) acetic acid in water (60 : 40 to 80 : 20, linear over 2 min), and for Mix 2, it was methanol : water (60 : 40 for 4 min, then ramped linearly to 90 : 10, over 12 min) pumped at 1.5 mL/min for 4 min, then 1 mL/min till the end of run. EXE, RES, LUT and flutamide (internal standard (IS)) were measured at 246, 307, 350 and 300 nm, respectively. For Mix 1, RES, EXE and IS eluted at 3.5, 6.8 and 7.4 min, respectively, while for Mix 2, LUT, EXE and IS eluted at 7.5, 11.4 and 12.7 min, respectively. The mean r(2) for the standard curves was ≥0.99, and percentage coefficient of variation and % error of the mean were <2. Both assays successfully quantitated Mix 1 and Mix 2 in their nanoformulations. The two developed assays were sensitive and selective for the analysis of EXE-LUT and EXE-RES mixtures in nanoformulations according to International Conference on Harmonization guidelines.

Metabolism of [17-2H]pregnenolone into 5-[17 beta-2H, 17 alpha-18O]androstene-3 beta, 17 alpha-diol and other products by incubation with the microsomal fraction of boar testis under 18O2 atmosphere.

[17-2H]Pregnenolone was incubated with the microsomal fraction of boar testis under an 18O2 atmosphere. The metabolites were analyzed by gas chromatography-mass spectrometry, and the following six metabolites labeled with 2H or 18O (or both) were identified: 17 alpha-[17-18O]hydroxypregnenolone, [17-18O]dehydroepiandrosterone, 5-[17-18O]androstene-3 beta, 17 beta-diol, 16 alpha-[16-18O]hydroxy[17-2H]pregnenolone, 5-[17 beta-2H, 17-18O]androstene-3 beta,17 alpha-diol, and 5,16-[17-2H]androstadien-3 beta-ol. The time course of the formation of these metabolites from pregnenolone was also studied using 14C-labeled substrate. The results obtained from these experiments suggest that the first three metabolites were synthesized by a well-documented pathway--pregnenolone yields 17 alpha-hydroxypregnenolone yields dehydroepiandrosterone yields 5-androstene-3 beta,17 beta-diol--, and that 16 alpha-hydroxypregnenolone, 5-androstene-3 beta,17 alpha-diol and 5,16-androstadien-3 beta-ol were synthesized from [17-2H]pregnenolone with retention of 17-2H.

Mass spectrometric analysis of androstan-17beta-ol-3-one and androstadiene-17beta-ol-3-one isomers.

Mass spectrometric identification and characterization of steroids using electrospray ionization and tandem mass spectrometry has advantages in drug testing and doping control analysis attributable to limitations of gas chromatography followed by electron ionization mass spectrometry. Steroids with an androstadiene-17beta-ol-3-one nucleus and double bonds located either at C-1 and C-4, C-4 and C-9, or C-4 and C-6 were used to determine characteristic fragmentation pathways. Diagnostic dissociation routes are proposed using deuterium labeling, MS3 experiments, and analyses of structurally closely related compounds. Steroids such as boldenone (androst-1,4-diene-17beta-ol-3-one) produced characteristic product ions at m/z 121, 135, and 147. Compounds with double bonds at C-4 and C-9 generated abundant product ions at m/z 145 and 147. Conjugated double bonds at C-4 and C-6 gave rise to an intense and characteristic signal at m/z 133. Stereochemical differentiation between 5alpha- and 5beta-isomers of androstan-17beta-ol-3-ones was possible because of significant differences in relative abundance of product ions generated by elimination of acetone from alpha,beta-saturated 3-keto steroids.

Andrastins A-D, Penicillium roqueforti Metabolites consistently produced in blue-mold-ripened cheese.

This is the first finding of andrastins in blue cheese as well as any other sample type. Here, they were produced by the secondary starter culture Penicillium roqueforti. After purification by normal-phase chromatography followed by combined reverse-phase ion-exchange chromatography, the andrastins A-D were detected by liquid chromatography combined with UV and high-resolution mass spectrometry. In 23 representative samples of European blue cheeses, andrastin A was consistently found in quantities between 0.1 and 3.7 microg/g of cheese (median 2.4 microg/g). Assuming the same molar response factors as for andrastin A, the B, C, and D analogues were present in approximately 5-, 3-, and 5-20-fold lower amounts than andrastin A, respectively. The andrastins are protein farnesyltransferase inhibitors and are capable of inhibiting the efflux of anticancer drugs from multidrug-resistant cancer cells. Thus, their presence in common blue cheese suggests a potential for a positive or negative impact on human health.

High-pressure aerosol suspensions--a novel laser diffraction particle sizing system for hydrofluoroalkane pressurised metered dose inhalers.

In this study, a novel laser diffraction particle size analysis dispersion system, capable of sizing particles in situ within suspension hydrofluoroalkane (HFA) pressurised metered dose inhalers (pMDIs), was developed and tested. The technique was compared to four indirect particle sizing methods commonly used to determine the size of particles suspended in HFA pMDIs. The median volume diameter obtained using laser diffraction of both the salbutamol sulphate and fluticasone propionate suspended either in 2H, 3H-decafluoropentane or perfluoropentane (employed as surrogate propellants) was over one-order of magnitude larger than the particle sizes of the drugs suspended in HFA 134a. In contrast, the "in-flight" particle size using the Sympatec inhaler 2000 laser diffraction equipment undersized the particles, predicting higher delivery efficacy compared to the other sizing methods. However, the size of particles suspended in HFAs derived using the novel pressurised dispersion system, showed a linear correlation with the impaction results, r2=0.8894 (n=10). The novel pressure cell sizing technique proved to be simple to use, has the ability to be automated and was accurate, suggesting it could be an essential tool in the development of new suspension-based pMDI formulations.