Dispersive liquid-liquid microextraction followed by sweeping micellar electrokinetic chromatography-tandem mass spectrometry for determination of six breast cancer drugs in human plasma.

Herein, we have developed a novel method of aqueous-sample dispersive liquid-liquid microextraction (AqS-DLLME) followed by sweeping micellar electrokinetic chromatography-tandem mass spectrometry (MEKC-MS/MS) for simultaneous determination of breast cancer drugs letrozole, anastrozole, palbociclib, ribociclib, abemaciclib, and fulvestrant in human plasma. Coupling of MEKC to MS was possible due to the use of ammonium perfluorooctanoate (APFO) as a volatile surfactant. The MEKC and MS conditions were optimized to achieve a fast, sensitive, selective, and green analysis enabling full separation of the analytes within 16 min. Electrophoretic buffer was 125 mM APFO at apparent pH 10.5 in 32 % MeOH, while sheath liquid was 70 % MeOH with 0.2 % formic acid, delivered at 10 µL/min. Excellent extraction recoveries from plasma ranging from 89.4 to 104.9 % were obtained with a combination of protein precipitation and DLLME. The developed method was validated according to the ICH guidelines. Remarkable selectivity, accuracy (bias < 6.7 %), precision (RSD < 15.8 %), and stability (bias < 10.4 %) with insignificant matrix effect (RSD < 14.0 %) and no carry-over were obtained over a wide range of concentrations. Linearity with inter-day slope RSD lower than 8.7 % was demonstrated. With this method, very low concentrations could be detected after the injection of only 68.7 nL of the sample. The method was applied to plasma samples from six women currently receiving breast cancer treatment. Determined concentrations of the drugs of interest agreed with concentrations found in clinical studies, thus proving the suitability of the developed method for therapeutic drug monitoring as a superior alternative to published LC-MS methods.

Simultaneous determination of macrolides in water samples by solid-phase extraction and capillary electrophoresis.

Solid-phase extraction (SPE) coupled with capillary electrophoresis (CE) for the determination of macrolide antibiotics (azithromycin, clarithromycin, roxithromycin, tylosin) and tiamulin in water samples was described in this article. These compounds were extracted with different types of sorbents ( Oasis HLB, C18, C8, SDB, and Strata-X) and different masses of sorbents (60 mg, 200 mg, and 500 mg) using different organic solvents (methanol, ethanol, and acetonitrile) and different pH values of water samples (pH 7.00, 8.00, and 9.00). It was found that the highest extraction efficiency of the studied compounds was obtained with 200 mg/3 mL C18 cartridges with methanol as eluent at pH 9.00 of the water sample. The developed SPE-CE method for macrolide antibiotics and tiamulin was validated for linearity, precision, repeatability, the limit of detection (LOD), the limit of quantification (LOQ), and recovery. Good linearity was obtained in the range of 0.3-30 mg L-1 depending on the drug, with correlation coefficients higher than 0.9958 in all cases except clarithromycin (0.9873). Expanded measurement uncertainties were calculated for each pharmaceutical, accounting for 20.31 % (azithromycin), 38.33 % (tiamulin), 28.95 % (clarithromycin), 26.99 % (roxithromycin), and 21.09 % (tiamulin). Uncertainties associated with precision and calibration curves contributed the most to the combined measurement uncertainty. The method was successfully applied to the analysis of production waste-water from the pharmaceutical industry.

Exploring adherence in patients with advanced breast cancer: focus on CDK4/6 inhibitors.

Treatment adherence is crucial for optimal outcomes in advanced breast cancer, but can be challenging due to various factors, i.e. patients' attitudes and behavior upon diagnosis, and complex therapies with high adverse effect rates. Our aim was to explore the adherence to oral anticancer medications (OAM) in women with advanced breast cancer, focusing on cyclin-dependent kinase 4 and 6 inhibitors (CDKI), and identify factors associated with the adherence. We conducted a cross-sectional study at the University Hospital Centre Zagreb, Croatia, involving women with stage IV advanced breast cancer receiving OAM. Data collection included a questionnaire assessing socio-demographic and clinical information, Beck Depression Inventory-II for depressive symptoms, Medication Adherence Report Scale (MARS-5) for adherence to OAM, and Beliefs about Medicines Questionnaire. Plasma concentrations of CDKI were confirmed by LC-MS/MS in three randomly selected participants. A total of 89 women were included. The most prescribed OAMs were anti-estrogen (71.3 %) and CDKI (60.9 %). MARS-5 scores (mean: 24.1 ± 1.6) correlated with CDKI plasma concentrations. Forgetfulness was the primary reason for non-adherence (25.9 %). Women receiving CDKI (p = 0.018), without depressive symptomatology (p = 0.043), and with more positive beliefs about medicines were more adherent (p < 0.05). This study enhances understanding of medication adherence in advanced breast cancer and identifies influential factors.

Optimisation of Solid-Phase Extraction and LC-MS/MS Analysis of Six Breast Cancer Drugs in Patient Plasma Samples.

In the development of bioanalytical LC-MS methods for the determination of drugs in plasma samples in a clinical setting, adequate sample preparation is of utmost importance. The main goals are to achieve the selective extraction of the analytes of interest and attain thorough matrix removal while retaining acceptable ecological properties, cost-effectiveness, and high throughput. Solid-phase extraction (SPE) offers a versatile range of options, from the selection of an appropriate sorbent to the optimisation of the washing and elution conditions. In this work, the first SPE method for the simultaneous extraction of six anticancer drugs used in novel therapeutic combinations for advanced breast cancer treatment-palbociclib, ribociclib, abemaciclib, anastrozole, letrozole, and fulvestrant-was developed. The following sorbent chemistries were tested: octylsilyl (C8), octadecylsilyl (C18), hydrophilic-lipophilic balance (HLB), mixed-mode cation-exchange (MCX and X-C), and mixed-mode weak cation-exchange (WCX), with different corresponding elution solvents. The samples were analysed using LC-MS/MS, with a phenyl column (150 × 4.6 mm, 2.5 µm). The best extraction recoveries (≥92.3%) of all analytes were obtained with the C8 phase, using methanol as the elution solvent. The optimised method was validated in the clinically relevant ranges, showing adequate precision (inter-day RSD ≤ 14.3%) and accuracy (inter-day bias -12.7-13.5%). Finally, its applicability was successfully proven by the analysis of samples from breast cancer patients.

Optimisation of dispersive liquid-liquid microextraction for plasma sample preparation in bioanalysis of CDK4/6 inhibitors in therapeutic combinations for breast cancer treatment.

Cyclin D dependent kinase 4 and 6 (CDK 4/6) inhibitors are novel anticancer drugs used in therapeutic combinations with endocrine therapy for breast cancer treatment. Their determination in patient plasma is of high interest as a prerequisite for possible therapeutic drug monitoring. Dispersive liquid-liquid microextraction (DLLME) shows great potential in bioanalytical sample preparation. Its simplicity and speed, along with the suitability for using small amounts of sample and hazardous solvents are some of its main advantages. However, its application on plasma samples is scarce and requires further development. The aim of this work was to explore the applicability of DLLME in the simultaneous extraction of six drugs of interest from human plasma, with an emphasis placed on achieving high extraction recoveries with low sample and solvent consumption. To tackle the low availability and amount of the plasma sample, as well as the complexity of the biological matrix, three novel DLLME modes are proposed: organic sample DLLME (OrS-DLLME), aqueous sample DLLME (AqS-DLLME), and a modified air-assisted DLLME (AA-DLLME). The extractant and disperser type and volume, volume ratios of all the components in the ternary system, effect of pH and salting out were optimised for all three proposed modes of DLLME. Optimised representative DLLME-HPLC-DAD-FLD method was validated and shown to be linear (R > 0.994), precise (RSD ≤13.8%, interday), accurate (bias -13.1-13.1%, interday) and robust (relative effect -3.34-6.08%). Simultaneous extraction of all six drugs with high recoveries (81.65-95.58%) was achieved. Sample volumes used were as low as 50-100 µL, with necessary organic solvent volumes in µL ranges. Greenness scores obtained using the AGREE software were between 0.63 and 0.66, demonstrating compliance with green analytical chemistry principles. Finally, the validated method was successfully applied on breast cancer patient plasma samples.

Rapid Capillary Electrophoresis Method for Simultaneous Determination of Abemaciclib, Ribociclib, and Palbociclib in Pharmaceutical Dosage Forms: A Green Approach.

Advances in the treatment of HR+/HER2- breast cancer phenotype have been made with the introduction of abemaciclib, ribociclib, and palbociclib, inhibitors of cyclin D dependent kinases 4 and 6 (CDK4/6). Here, a novel, fast, cheap, and green CE method for the simultaneous determination of these three CDK4/6 inhibitors in less than 4 min is proposed for the first time. Separation was achieved by capillary zone electrophoresis in an acidic medium, in accordance with the structures of the analytes and their pKa values. The optimal pH of the running buffer was found to be 2.9. The optimal method conditions were 27.5 kV separation voltage, 30 °C, 5 s injection time under 50 mbar pressure, and 50 mM phosphate background buffer with benzimidazole as an internal standard. The developed method was validated with respect to robustness, selectivity, accuracy, precision, linearity, and limits of detection. The method was shown to be linear in the range of 10 to 100 µg mL-1 with correlation coefficients higher than 0.9981. A greenness assessment of the proposed method was performed, and the method was shown to be green. The validated method was successfully applied to pharmaceutical dosage forms of all CDK4/6 inhibitors.

Development and Validation of a Novel LC-MS/MS Method for the Simultaneous Determination of Abemaciclib, Palbociclib, Ribociclib, Anastrozole, Letrozole, and Fulvestrant in Plasma Samples: A Prerequisite for Personalized Breast Cancer Treatment.

Palbociclib, ribociclib and abemaciclib were recently approved as chemotherapeutic agents and are currently in the post-marketing surveillance phase. They are used in combination with aromatase inhibitors anastrozole and letrozole or antiestrogen fulvestrant for HR+, HER2- breast cancer treatment. Here, a novel bioanalytical LC-ESI-MS/MS method was developed for the quantitation of these six drugs in human plasma. The samples were prepared by simple protein precipitation followed by solvent evaporation. A Kinetex biphenyl column (150 × 4.6 mm, 2.6 µm) used for chromatographic analysis adequately resolved even the closely eluting aromatase inhibitors' peaks. The mobile phase consisted of 0.1% formic acid in water and in ACN, in a linear gradient. An additional gradient step was added to eliminate the observed carry-over. The proposed method was fully validated in the relevant linear ranges covering the expected plasma concentrations of all six drugs (correlation coefficients between 0.9996 and 0.9931). The intra-day method precision (CV) ranged from 3.1% to 15%, while intra-day accuracy (%bias) was between -1.5% and 15.0%. The inter-day precision ranged from 1.6% to 14.9%, with accuracy between -14.3% and 14.6%, which is in accordance with the EMA and ICH guidelines on bioanalytical method validation. The method was successfully applied to samples from patients treated for HR+, HER2- breast cancer.

Development and Validation of an HPLC Method for Simultaneous Determination of Capsaicinoids and Camphor in Over-the-Counter Medication for Topical Use.

A reverse-phase high-performance liquid chromatography method was developed to determine and quantify capsaicin (trans-8-methyl-N-vanillyl-6- nonenamid), dihydrocapsaicin (8-methyl-N-vanillylnonanamide), and camphor (trimethylbicyclo[2.2.1]heptan-2-one). It is applicable in analyses of over-the-counter (OTC) medications for topical use and raw materials such as chili pepper oleoresin. Chromatographic separation was carried out on a C18 column using an isocratic flow of the mobile phase containing acetonitrile and ultrapure water in a ratio of 2:3, with pH adjusted to 3.2 using glacial acetic acid, and a flow rate of 1.5 mL/min. The concentration of the eluting compounds was monitored by a diode-array detector at a wavelength of 281 nm. The method was evaluated for several validation parameters, including selectivity, accuracy (confidence intervals < 0.05%), repeatability, and intermediate precision. The limit of detection (LOD) was determined to be 0.070 µg/mL for capsaicin, 0.211 µg/mL for dihydrocapsaicin, and 0.060 µg/mL for camphor. The limit of quantification (LOQ) was determined to be 0.212 µg/mL for capsaicin, 0.640 µg/mL for dihydrocapsaicin, and 0.320 µg/mL for camphor. Linearity was set in the range of 2.5-200 µg/mL for capsaicin and dihydrocapsaicin and 25-2000 µg/mL for camphor. The suggested analytical method can be used for quality control of formulated pharmaceutical products containing capsaicinoids, camphor, and propolis.

Fungi and their secondary metabolites in water-damaged indoors after a major flood event in eastern Croatia.

In winter and summer of 2016 and 2017, airborne fungi and house dust were collected in indoors of the village Gunja, which had been flooded, and the control village Gornji Stupnik (Croatia) in order to explore variations of fungal indoor levels, particularly Aspergilli section Nidulantes series Versicolores, as well as fungal metabolites in dust. Levels of airborne Aspergilli (Versicolores) were three times as high in winter and summer in Gunja than in the control village, while dustborne isolates were equally present in both locations. Sequencing of the calmodulin gene region revealed that among Aspergilli (Versicolores), A. jensenii and A. creber were dominant and together with A. puulaauensis, A. tennesseensis and A. venenatus produced sterigmatocystin and 5-methoxysterigmatocystin (HPLC coupled with mass spectrometry); A. amoenus, A. fructus, A. griseoaurantiacus, A. pepii, and A. protuberus produced sterigmatocystin but not 5-methoxysterigmatocystin; A. sydowii did not produce any of these toxins. A total of 75 metabolites related to Penicillium (29), Aspergillus (22), Fusarium (10), Alternaria (5), Stachybotrys (2), and other fungi (7) were detected in dust by liquid chromatography-tandem mass spectrometry. The majority of metabolites including sterigmatocystin and 5-methoxysterigmatocystin exhibited a higher prevalence in winter in Gunja.

Post-Flood Impacts on Occurrence and Distribution of Mycotoxin-Producing Aspergilli from the Sections Circumdati, Flavi, and Nigri in Indoor Environment.

Mycotoxin-producing Aspergilli (Circumdati, Flavi, and Nigri), usually associated with contaminated food, may also cause respiratory disorders and are insufficiently studied in water-damaged indoor environments. Airborne (N = 71) and dust borne (N = 76) Aspergilli collected at post-flood and control locations in Croatia resulted in eleven different species based on their calmodulin marker: A. ochraceus, A. ostianus, A. pallidofulvus, A. sclerotiorum, and A. westerdijkiae (Circumdati); A. flavus (Flavi); and A. tubingensis, A. welwitschiae, A. niger, A. piperis, and A. uvarum (Nigri). Most of the airborne (73%) and dust borne (54%) isolates were found at post-flood locations, and the highest concentrations measured in indoor air (5720 colony-forming units (CFU)/m3) and dust (2.5 × 105 CFU/g) were up to twenty times higher than in the control locations. A. flavus dominated among airborne isolates (25%) at the unrepaired locations, while 56% of the dust borne Aspergilli were identified as A. tubingensis and A. welwitschiae. The ability of identified isolates to produce mycotoxins aflatoxin B1 (AFB1), fumonisin B2 (FB2), and ochratoxin A were assessed by LC-MS analysis. All ochratoxin A (OTA)-producing Circumdati belonged to A. westerdijkiae (13.7 ± 15.81 µg/mL); in the section, FlaviA. flavus produced AFB1 (2.51 ± 5.31 µg/mL), while A. welwitschiae and A. niger (section Nigri) produced FB2 (6.76 ± 13.51 µg/mL and 11.24 ± 18.30 µg/mL, respectively). Water damage dominantly supported the occurrence of aflatoxigenic A. flavus in indoor environments. Yet unresolved, the causal relationship of exposure to indoor Aspergilli and adverse health effects may support the significance of this research.

Evaluation of alcohol content and metal impurities in liquid dietary supplements by sHSS-GC-FID and GFAAS techniques.

Despite efforts by many dietary supplements' manufactures to reduce or replace ethanol, many products containing ethanol in concentrations up to 70% are available on market. Furthermore, botanical dietary supplements can vary in metal content as a function of the environment, processing equipment and product containers. Therefore, the aim of study was to develop a new rapid and highly sensitive method for simultaneous determination of ethanol and its impurities in dietary supplements by sHSS-GC-FID technique. In addition, contamination with metals by GFAAS technique was evaluated. The proposed sHSS-GC-FID method was successfully applied for analysis of 93 samples containing various amounts of ethanol. It should be highlighted that the dramatic variation from manufacture's claims was found in even one third of products. Furthermore, high amounts of ethanol were found in several products especially designed for children and in one product labeled as "alcohol-free". Metal impurities were below the limits established by USP.

Multi-walled carbon nanotubes/Nafion composite film modified electrode as a sensor for simultaneous determination of ondansetron and morphine.

The electrochemical behavior of ondansetron was studied on the multi-walled carbon nanotubes/Nafion polymer composite modified glassy carbon electrode (MWCNTs-Nafion/GCE). The oxidation peak potential was shifted from 1.32 V to 1.18 V compared to the bare electrode indicating excellent electrocatalytic activity of immobilized film toward drug molecule. The modified electrode exhibited a remarkable enhancement effect on voltammetric response due to the synergistic effect of nanomaterial and cation-exchange polymer on the electron transfer rate, the effective electrode area and the accumulation capability. After optimizing the experimental parameters, adsorptive stripping procedure was used for the determination of ondansetron in pharmaceutical formulation. The results were satisfactory in comparison with those obtained by high-performance liquid chromatography. In addition, the MWCNTs-Nafion/GCE exhibited high selectivity in the voltammetric measurements of ondansetron and co-administrated drug morphine with potential difference of 430 mV. The response peak currents had linear relationship with drug concentration in the range of 1.0 × 10(-7)-5.0 × 10(-6)M and 1.0 × 10(-7)-4.0 × 10(-6)M with detection limits 3.1 × 10(-8) and 3.2 × 10(-8)M for ondansetron and morphine, respectively. The electrode was successfully applied for simultaneous electrochemical sensing of both drugs in human serum samples after selective accumulation at the electrode surface.

Development of a rapid LC/DAD/FLD/MS(n) method for the simultaneous determination of monacolins and citrinin in red fermented rice products.

Red fermented rice is used worldwide by many patients as an alternative therapy for hyperlipidemia; however, the discovery of a toxic fermentation byproduct, citrinin, causes much controversy about the safety of red mold rice products. A new and fast high-performance liquid chromatography method was developed and validated for simultaneous determination of cholesterol-lowering compounds monacolin K (lovastatin), monacolin K hydroxy acid, and other monacolins present in red fermented rice as well as nephrotoxic mycotoxin citrinin in a single run using connected diode array and fluorescence and mass spectrometric detectors. The proposed method was successfully applied for the analysis of red fermented rice food samples and various dietary supplements also containing other natural lipid-lowering agents. The deviations between label content and levels of active compounds found in investigated samples as well as high batch-to-batch variation found in one product indicate that the regular quality control of red fermented rice products is of great importance.

Simultaneous determination of lovastatin and citrinin in red yeast rice supplements by micellar electrokinetic capillary chromatography.

Lovastatin is a main component of Monascus purpureus fermented red rice contributing to the lipid-lowering effect. Citrinin is a toxic fermentation by-product which can be found as a contaminant. An accurate, simple and rapid micellar electrokinetic capillary chromatographic method was developed for the first time for simultaneous determination of lovastatin present in lactone and hydroxy acid forms and citrinin in red rice products provided by different manufacturers and formulated in various dosage forms. Separation was achieved within only 2 min using 20 mM of phosphate buffer at pH 7.0 and 30 mM of sodium dodecyl sulphate at an applied voltage of 25 kV. Sensitivity crucial for detecting citrinin was enhanced by using an extended light path capillary. The results showed that the content of lovastatin and its acid form in dietary supplements were considerably different indicating the need for improved standardization in order to ensure efficiency and safety of these products.

Simultaneous analysis of mitotane and its main metabolites in human blood and urine samples by SPE-HPLC technique.

Adrenocortical carcinoma (ACC) is a rare malignancy with an incompletely understood pathogenesis and a poor prognosis. The adrenalytic activity of mitotane has made it the most important single drug in the treatment of ACC. Unfortunately, the exact mechanism of mitotane action is still unknown. It is believed that mitotane belongs to the class of drugs that require metabolic transformation by cytochrome P450 for therapeutic action; therefore determination of plasma levels of not only mitotane but also its metabolites would help in carrying out the treatment. The objective of this work was to develop and validate an SPE-HPLC method for simultaneous determination of mitotane and its metabolites in different biological fluids. The sample preparation consisted of a solid-phase extraction on a Discovery DSC(18) cartridge, while analysis of extracts was performed on a Symmetry C(18) column. The usefulness of the proposed method was confirmed by analysis of plasma, red cell and urine samples from patient chronically treated with 1.5 g of mitotane. The patient involved in this study had a high plasma concentration of mitotane and none of the investigated metabolites were found. In order to investigate whether the polymorphism of CYP2C9 and CYP2C19 enzymes could be related to the metabolism of mitotane, RT-PCR analysis was performed.