Wipe sampling procedure coupled to LC-MS/MS analysis for the simultaneous determination of 10 cytotoxic drugs on different surfaces

A simple wipe sampling procedure was developed for the surface contamination determination of ten cytotoxic drugs: cytarabine, gemcitabine, methotrexate, etoposide phosphate, cyclophosphamide, ifosfamide, irinotecan, doxorubicin, epirubicin and vincristine. Wiping was performed using Whatman filter paper on different surfaces such as stainless steel, polypropylene, polystyrol, glass, latex gloves, computer mouse and coated paperboard. Wiping and desorption procedures were investigated: The same solution containing 20% acetonitrile and 0.1% formic acid in water gave the best results. After ultrasonic desorption and then centrifugation, samples were analysed by a validated liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in selected reaction monitoring mode. The whole analytical strategy from wipe sampling to LC-MS/MS analysis was evaluated to determine quantitative performance. The lowest limit of quantification of 10ng per wiping sample (i.e. 0.1ngcm−2) was determined for the ten investigated cytotoxic drugs. Relative standard deviation for intermediate precision was always inferior to 20%. As recovery was dependent on the tested surface for each drug, a correction factor was determined and applied for real samples. The method was then successfully applied at the cytotoxic production unit of the Geneva University Hospitals pharmacy. Figure Wipe sampling procedure for the determination of cytotoxic drug

Simultaneous quantification of ten cytotoxic drugs by a validated LC-ESI-MS/MS method

A liquid chromatography separation with electrospray ionisation and tandem mass spectrometry detection method was developed for the simultaneous quantification of ten commonly handled cytotoxic drugs in a hospital pharmacy. These cytotoxic drugs are cytarabine, gemcitabine, methotrexate, etoposide phosphate, cyclophosphamide, ifosfamide, irinotecan, doxorubicin, epirubicin and vincristine. The chromatographic separation was carried out by RPLC in less than 21min, applying a gradient elution of water and acetonitrile in the presence of 0.1% formic acid. MS/MS was performed on a triple quadrupole in selected reaction monitoring mode. The analytical method was validated to determine the limit of quantification (LOQ) and quantitative performance: lowest LOQs were between 0.25 and 2ngmL−1 for the ten investigated cytotoxic drugs; trueness values (i.e. recovery) were between 85% and 110%, and relative standard deviations for both repeatability and intermediate precision were always inferior to 15%. The multi-compound method was successfully applied for the quality control of pharmaceutical formulations and for analyses of spiked samples on potentially contaminated surfaces. Figure Preparation of cytotoxic formulations at the Pharmacy of Geneva University Hospital

Comparison of Healthy and Dandruff Scalp Microbiome Reveals the Role of Commensals in Scalp Health

Several scalp microbiome studies from different populations have revealed the association of dandruff with bacterial and fungal dysbiosis. However, the functional role of scalp microbiota in scalp disorders and health remains scarcely explored. Here, we examined the bacterial and fungal diversity of the scalp microbiome and their potential functional role in the healthy and dandruff scalp of 140 Indian women. Propionibacterium acnes and Staphylococcus epidermidis emerged as the core bacterial species, where the former was associated with a healthy scalp and the latter with dandruff scalp. Along with the commonly occurring Malassezia species (M. restricta and M. globosa) on the scalp, a strikingly high association of dandruff with yet uncharacterized Malassezia species was observed in the core mycobiome. Functional analysis showed that the fungal microbiome was enriched in pathways majorly implicated in cell-host adhesion in the dandruff scalp, while the bacterial microbiome showed a conspicuous enrichment of pathways related to the synthesis and metabolism of amino acids, biotin, and other B-vitamins, which are reported as essential nutrients for hair growth. A systematic measurement of scalp clinical and physiological parameters was also carried out, which showed significant correlations with the microbiome and their associated functional pathways. The results point toward a new potential role of bacterial commensals in maintaining the scalp nutrient homoeostasis and highlights an important and yet unknown role of the scalp microbiome, similar to the gut microbiome. This study, therefore, provides new perspectives on the better understanding of the pathophysiology of dandruff

Use of monolithic supports for fast analysis of drugs and metabolites in plasma by direct injection

Commercial monolithic columns were evcluated as extraction supports for the analysis of various drugs and their metabolites from plasma. For this purpose, a precolumn packed with a monolithic phase was used as extraction and enrichment support and integrated in a column-switching system for the direct and simultaneous analysis of fluoxetine and norfluoxetine, methadone and EDDP, flunitrazepam and norflunitrazepom in humon plasma. In this configuration, plasma samples were directly injected onto a Chromolith Flash precolumn (25×4.6 mm I.D.) coupled to a microbore analytical column packed with a conventional reversed-phase malerial. Detection was by an electrospray-ionization mass spectrometer (ESl-MS). Total analysis time, including extraction and separation of the six analytes, was <10 min. For all the investigated compounds, the limits of quantitation (LOQ) were estimated in the ng.mL1 range for an injection volume of 50 μL. The method was also applied to a real clinical case to demonstrate its applicability

New extraction supports for the fast analysis of pharmaceutical compounds in biological samples by direct injection

The number of samples to be analyzed during the drug discovery process is constantly increasing. Therefore, fast, selective and sensitive analytical methods are required. Nowadays, liquid chromatography coupled with mass spectrometry (LC-MS) is considered as the method of choice. With this performant tool, the sample preparation step prior to analysis can be simplified and the analytical process speeded up. Therefore, several sample extraction techniques, coupled on-line with a LC-MS system, have recently been developed. In this work, different extraction supports allowing the direct injection of biological fluids were investigated. In the single column mode or in the column switching configuration, these supports were found suitable for the fast analysis (less than 15 min) of drugs and their metabolites present at ng.ml-1 level in biological matrices

Matrix effect in LC-ESI-MS and LC-APCI-MS with off-line and on-line extraction procedures

Matrix effect on mass spectrometry response was investigated with commercially available electrospray ionisation (ESI) and atmospheric pressure chemical ionisation (APCI) sources coupled with a single quadruple mass spectrometer. A post-column infusion system was used to observe the MS signal alterations of methadone, selected as the model compound, in plasma. For this purpose, two sample preparation procedures were tested: (1) conventional off-line sample preparations with liquid-liquid extraction (LLE), solid-phase extraction (SPE) and protein precipitation (PP) with perchloric acid (PA) and acetonitrile (ACN) and (2) on-line SPE with two different extraction columns packed with a large particle support (LPS) and with restricted access material (RAM), respectively. Whatever the sample preparation procedures, APCI source appeared to be less liable to matrix effect than ESI source. Among the different off-line sample preparations, LLE was the most efficient extraction procedure. With other techniques, MS signal was affected not only by endogenous material but also by procedure introduced compounds. Moreover, on-line SPE-LC-MS configuration exhibited matrix effects, which depend on the API source and the extraction support

Restricted access materials and large particle supports for on-line sample preparation: an attractive approach for biological fluids analysis

An analytical process generally involves four main steps: (1) sample preparation; (2) analytical separation; (3) detection; and (4) data handling. In the bioanalytical field, sample preparation is often considered as the time-limiting step. Indeed, the extraction techniques commonly used for biological matrices such as liquid-liquid extraction (LLE) and solid-phase extraction (SPE) are achieved in the off-line mode. In order to perform a high throughput analysis, efforts have been engaged in developing a faster sample purification process. Among different strategies, the introduction of special extraction sorbents, such as the restricted access media (RAM) and large particle supports (LPS), allowing the direct and repetitive injection of complex biological matrices, represents a very attractive approach. Integrated in a liquid chromatography (LC) system, these extraction supports lead to the automation, simplification and speeding up of the sample preparation process. In this paper, RAM and LPS are reviewed and particular attention is given to commercially available supports. Applications of these extraction supports, are presented in single column and column-switching configurations, for the direct analysis of compounds in various biological fluids

Column-switching procedure for the fast analysis of drugs in biological samples

Liquid chromatography coupled with mass spectrometry in single and dual mode (LC-MS and LC-MS/MS) is the method of choice for the quantification of drugs and their metabolites in biologic fluids. Following the new challenges encountered in the process of drug development, liquid chromatography-mass spectrometry has been found to achieve high-throughput analysis. With this impressive tool, the sample preparation step before analysis is simplified, and the analytic process speeded up. Several generic approaches have recently been developed for the sample extraction coupled on line with a LC-MS system. In this paper, different extraction supports allowing the direct injection of biologic fluids were investigated, namely, restricted- access media, large-size particle, and monolithic phases. In the column-switching configuration, these supports, coupled with microbore analytic columns, were found suitable for the fast analysis (total analysis time of less than 10 minutes) of different drugs and their metabolites in biologic matrices at the nanogram per milliliter level

Rapid analysis of fluoxetine and its metabolite in plasma by LC-MS with column-switching approach

A rapid and sensitive method was developed for the simultaneous determination of fluoxetine and its primary metabolite, norfluoxetine, in plasma. It was based on a column-switching approach with a precolumn packed with large size particles coupled with a liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS). After a simple centrifugation, plasma samples were directly injected onto the precolumn. The endogenous material was excluded thanks to a high flow rate while analytes were retained by hydrophobic interactions. Afterwards, the target compounds were eluted in back flush mode to an octadecyl analytical column and detected by ESI-MS. The overall analysis time per sample, from plasma sample preparation to data acquisition, was achieved in less than 4 min. Method performances were evaluated. The method showed good linearity in the range of 25-1000 ng mL(-1) with a determination coefficient higher than 0.99. Limits of quantification were estimated at 25 ng mL(-1) for fluoxetine and norfluoxetine. Moreover, method precision was better than 6% in the studied concentration range. These results demonstrated that the method could be used to quantify target compounds. Finally, the developed assay proved to be suitable for the simultaneous analysis of fluoxetine and its metabolite in real plasma samples