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

Abstract.: 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 4min. Method performances were evaluated. The method showed good linearity in the range of 25-1000ngmL−1 with a determination coefficient higher than 0.99. Limits of quantification were estimated at 25ngmL−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 sample

Efficacy of Two Cleaning Solutions for the Decontamination of 10 Antineoplastic Agents in the Biosafety Cabinets of a Hospital Pharmacy

OBJECTIVE: This study aimed to evaluate two cleaning solutions for the chemical decontamination of antineoplastic agents on the surfaces of two biosafety cabinets routinely used for chemotherapy preparation in a hospital pharmacy. METHODS: For almost 1 year (49 weeks), two different solutions were used for the weekly cleaning of two biosafety cabinets in a hospital pharmacy's centralized cytotoxic preparation unit. The solutions evaluated were a commercial solution of isopropyl alcohol (IPA) and water (70:30, vol:vol), and a detergent solution constituted by 10(-2)M of sodium dodecyl sulfate (SDS) with 20% IPA. Seven areas in each biosafety cabinet were wiped 14 times throughout the year, before and after the weekly cleaning process, according to a validated procedure. Samples were analyzed using a validated method of high-performance liquid chromatography coupled to mass spectrometry. The decontamination efficacy of these two solutions was tested for 10 antineoplastic agents: cytarabine, gemcitabine, methotrexate, etoposide phosphate, irinotecan, cyclophosphamide, ifosfamide, doxorubicin, epirubicin, and vincristine. RESULTS: Overall decontamination efficacies observed were 82±6% and 49±11% for SDS solution and IPA, respectively. Higher contamination levels were distributed on areas frequently touched by the pharmacy technicians-such as sleeves and airlock handles-than on scale plates, gravimetric control hardware, and work benches. Detected contaminations of cyclophosphamide, ifosfamide, gemcitabine, and cytarabine were higher than those of the others agents. SDS solution was almost 20% more efficient than IPA on eight of the antineoplastic agents. CONCLUSION: Both cleaning solutions were able to reduce contamination levels in the biosafety cabinets. The efficacy of the solution containing an anionic detergent agent (SDS) was shown to be generally higher than that of IPA and, after the SDS cleaning procedure, biosafety cabinets demonstrated acceptable contamination levels

Evaluation of decontamination efficacy of cleaning solutions on stainless steel and glass surfaces contaminated by 10 antineoplastic agents

OBJECTIVES: The handling of antineoplastic agents results in chronic surface contamination that must be minimized and eliminated. This study was designed to assess the potential of several chemical solutions to decontaminate two types of work surfaces that were intentionally contaminated with antineoplastic drugs. METHODS: A range of solutions with variable physicochemical properties such as their hydrophilic/hydrophobic balance, oxidizing power, desorption, and solubilization were tested: ultrapure water, isopropyl alcohol, acetone, sodium hypochlorite, and surfactants such as dishwashing liquid (DWL), sodium dodecyl sulfate (SDS), Tween 40, and Span 80. These solutions were tested on 10 antineoplastic drugs: cytarabine, gemcitabine, methotrexate, etoposide phosphate, irinotecan, cyclophosphamide, ifosfamide, doxorubicin, epirubicin, and vincristine. To simulate contaminated surfaces, these molecules (200ng) were deliberately spread onto two types of work surfaces: stainless steel and glass. Recovered by wiping with a specific aqueous solvent (acetonitrile/HCOOH; 20/0.1%) and an absorbent wipe (Whatman 903®), the residual contamination was quantified using high-performance liquid chromatography (HPLC) coupled to mass spectrometry. To compare all tested cleaning solutions, a performance value of effectiveness was determined from contamination residues of the 10 drugs. RESULTS: Sodium hypochlorite showed the highest overall effectiveness with 98% contamination removed. Ultrapure water, isopropyl alcohol/water, and acetone were less effective with effectiveness values of 76.8, 80.7, and 40.4%, respectively. Ultrapure water was effective on most hydrophilic molecules (97.1% for cytarabine), while on the other hand, isopropyl alcohol/water (70/30, vol/vol) was effective on the least hydrophilic ones (85.2% for doxorubicin and 87.8% for epirubicin). Acetone had little effect, whatever the type of molecule. Among products containing surfactants, DWL was found effective (91.5%), but its formulation was unknown. Formulations with single surfactant non-ionics (tween 40 and span 80) or anionic (SDS) were also tested. Finally, solutions containing 10(-2) M anionic surfactants and 20% isopropyl alcohol had the highest global effectiveness at around 90%. More precisely, their efficacy was the highest (94.8%) for the most hydrophilic compounds such as cytarabine and around 80.0% for anthracyclines. Finally, the addition of isopropyl alcohol to surfactant solutions enhanced their decontamination efficiency on the least hydrophilic molecules. Measured values from the stainless steel surface were similar to those from the glass one. CONCLUSION: This study demonstrates that all decontamination agents reduce antineoplastic contamination on work surfaces, but none removes it totally. Although very effective, sodium hypochlorite cannot be used routinely on stainless steel surfaces. Solutions containing anionic surfactant such as SDS, with a high efficiency/safety ratio, proved most promising in terms of surface decontamination

Development of an online SPE–LC–MS-based assay using endogenous substrate for investigation of soluble epoxide hydrolase (sEH) inhibitors

Soluble epoxide hydrolase (sEH) is a promising therapeutic target for the treatment of hypertension, pain, and inflammation-related diseases. In order to enable the development of sEH inhibitors (sEHIs), assays are needed for determination of their potency. Therefore, we developed a new method utilizing an epoxide of arachidonic acid (14(15)-EpETrE) as substrate. Incubation samples were directly injected without purification into an online solid phase extraction (SPE) liquid chromatography electrospray ionization tandem mass spectrometry (LC–ESI–MS–MS) setup allowing a total run time of only 108 s for a full gradient separation. Analytes were extracted from the matrix within 30 s by turbulent flow chromatography. Subsequently, a full gradient separation was carried out on a 50X2.1 mm RP-18 column filled with 1.7 μm core–shell particles. The analytes were detected with high sensitivity by ESI–MS–MS in SRM mode. The substrate 14(15)-EpETrE eluted at a stable retention time of 96 ± 1 s and its sEH hydrolysis product 14,15-DiHETrE at 63 ± 1 s with narrow peak width (full width at half maximum height: 1.5 ± 0.1 s). The analytical performance of the method was excellent, with a limit of detection of 2 fmol on column, a linear range of over three orders of magnitude, and a negligible carry-over of 0.1% for 14,15-DiHETrE. The enzyme assay was carried out in a 96-well plate format, and near perfect sigmoidal dose–response curves were obtained for 12 concentrations of each inhibitor in only 22 min, enabling precise determination of IC50 values. In contrast with other approaches, this method enables quantitative evaluation of potent sEHIs with picomolar potencies because only 33 pmol L−1 sEH were used in the reaction vessel. This was demonstrated by ranking ten compounds by their activity; in the fluorescence method all yielded IC50 ≤ 1 nmol L−1. Comparison of 13 inhibitors with IC50 values >1 nmol L−1 showed a good correlation with the fluorescence method (linear correlation coefficient 0.9, slope 0.95, Spearman’s rho 0.9). For individual compounds, however, up to eightfold differences in potencies between this and the fluorescence method were obtained. Therefore, enzyme assays using natural substrate, as described here, are indispensable for reliable determination of structure–activity relationships for sEH inhibition

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

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