Siegrun A.I. Mohring

Evaluation and subsequent minimization of matrix effects caused by phospholipids in LC-MS analysis of biological samples

BACKGROUND The influence of matrix effects in LC-MS/MS analysis of biological samples can be enormous and has to be evaluated during method development. Phospholipids, which are present in considerable quantities in biological fluids, are supposed to cause matrix effects when co-eluting with analytes. Therefore, the reduction of phospholipids should lead to the minimization of matrix effects. METHODOLOGY & RESULTS: Here, a polymeric reversed-phase (PRP) SPE cartridge was compared with a combination of mixed-mode-anion-exchange (MAX) and mixed-mode-cation-exchange (MCX) SPE cartridges regarding elimination of matrix effects during sample clean-up. For evaluation of matrix effects post-column infusion experiments were performed. Phospholipid amount in the sample extract and matrix effects are enhanced using PRP in contrast to the combination of MAX/MCX. CONCLUSION For an efficient elimination of phospholipids during sample preparation and to improve method accuracy and precision it is advisable to use a combination of MAX/MCX SPE cartridges.

Determination of fluoroquinolones in chicken feces - a new liquid-liquid extraction method combined with LC-MS/MS.

The application of antibiotics including fluoroquinolones to farming animals is widespread and may lead to the development of antibiotic resistance and other environmental effects. To calculate environmental loads and for a proper risk assessment it is necessary to determine the antibiotic concentration in feces. Therefore, a new liquid-liquid extraction method combined with HPLC-MS/MS for the detection of marbofloxacin, ciprofloxacin, enrofloxacin and difloxacin in chicken feces was developed. Recoveries ranged from 51.0% to 83.5%. LOQs were between 0.10 and 1.09μg/kg. Feces of chickens treated with an enrofloxacin dosage of 10mg/kg bodyweight revealed maximum enrofloxacin and ciprofloxacin concentrations of 61.3 and 18.8mg/kg. Both antibiotics could be detected in feces up to two days after the last application in notable amounts (∼1mg/kg). Thus, feces of recently medicated chickens should not be used as a fertilizer without any further processing.

Cytotoxic drug residues in urine of dogs receiving anticancer chemotherapy.

BACKGROUND The presence of cytotoxic drug residues in urine of dogs may represent an exposure risk for pet owners and other people as well as a potential environmental contaminant. However, studies on cytotoxic drug residues in excretions of clinical patients are lacking in veterinary oncology. HYPOTHESIS Variable concentrations of cytotoxic residues are present in urine samples, depending on sampling time and substance. ANIMALS Client-owned dogs with lymphoma or mast cell tumors treated with standard chemotherapy protocols. METHODS Urine samples were collected before, directly after, and on days after administration of chemotherapy. Measurement of vincristine, vinblastine, cyclophosphamide, and doxorubicin residues in canine urine was performed by a quantitative liquid chromatography tandem mass spectrometry (LC/MS/MS) method. RESULTS Median cyclophosphamide residue concentration was 398.2 microg/L directly after treatment (d0) and was below the level of detection on days 1-3 (d1, d2, d3). Median vincristine residue concentration was 53.8 microg/L directly after treatment and was 20.2, 11.4, and 6.6 microg/L on days 1, 2, and 3. Median vinblastine residues were 144.9 (d0), 70.8 (d1), 35.6 (d2), and 18.7 microg/L (d3) with low concentrations detectable for 7 days after treatment. Median urine doxorubicin concentrations were 354.0 (d0), 165.6 (d1), 156.9 (d2), and 158.2 microg/L (d3). Low concentrations of doxorubicin were measurable up to 21 days after administration. CONCLUSIONS AND CLINICAL IMPORTANCE Variable concentrations of chemotherapeutics were measured in urine samples, depending on sampling time point and drug. Findings may inform current chemoprotection guidelines and help minimize exposure risks.

Comparison of different solid-phase extraction materials for the determination of fluoroquinolones in chicken plasma by LC-MS/MS.

Fluoroquinolones are synthetic antibiotics which are frequently used in veterinary medicine e.g. for the treatment of poultry. Their specific importance is based on the fact that they are regarded as antibiotics of last resort because of their broad spectrum of action against Gram-negative and -positive bacteria. Here, a new and sensitive method for the simultaneous determination of four fluoroquinolones (marbofloxacin, ciprofloxacin, enrofloxacin and difloxacin) in chicken plasma by LC-MS/MS was developed. Solid-phase extraction was chosen for sample preparation because a selective sample clean-up is combined with an effective extraction. Various solid-phase extraction materials including polymer-based reversed-phase, silica-based reversed-phase and mixed-mode sorbents were compared. Selection criteria were analyte recovery, sample extract purity and economical aspects (analysis time and elution solvent volume). Best recoveries and minimized elution solvent volumes were achieved using polymeric reversed-phase cartridges. However, post-column infusion experiments revealed that the analysis is influenced by co-eluting matrix components. Hence, a combination of a mixed-mode anion-exchange cartridge and a mixed-mode cation-exchange cartridge was used as final extraction method. This method yield slightly lower analyte recoveries compared to polymeric-reversed-phase cartridges but exhibit no matrix effects. Recoveries of spiked chicken plasma ranged from 61.9% to 84.8% with an inter-day precision of generally less than 12%. LODs are between 0.03 and 0.05μg/L; LOQs are between 0.08 and 0.16μg/L. Maximum plasma concentrations of chickens medicated with an enrofloxacin dosage of 3mg/kg bodyweight were 38.9μg/L for enrofloxacin and 3.3μg/L for its main metabolite ciprofloxacin.

Biocompatibility and antibacterial activity of photolytic products of sulfonamides

BACKGROUND This study assessed the photochemical fate of nine sulfonamides (sulfamerazine, sulfanilamide, sulfamethoxypyridazine, sulfamethoxazole, sulfachloropyridazine, sulfamethazine, sulfadiazine, sulfathiazole and sulfadimethoxine) during a 6h irradiation period with UVA/UVB-light and UVA-light and over 7 days under natural (sunlight) conditions. The cell growth inhibition effect and cytotoxicity of sulfonamides and their photodegradation products was investigated over 24 and 48 h with murine fibroblasts and keratinocytes. Antibacterial activity of the degradation products was studied using the Geobacillus stearothermophilus var. Calidolactis C953 assay. RESULTS UVA/UVB treatment of several sulfonamide solutions results in degradation of the compounds in different amounts with the highest degradation rate for sulfathiazole and sulfanilamide. The UVA/UVB light degradation products exhibit no antimicrobial activity. Sun light exposure over 7 days reveals a similar degradation pattern of the different sulfonamides, albeit to a different extent. Compared with UVA/UVB-irradiation, UVA-irradiated sulfonamides degrade to a lesser extent (except sulfamethazine). There was no impact on cell toxicity of the UVA/UVB-degrading products except for sulfanilamide, while a slight impact on cell proliferation was observed. CONCLUSIONS All studied sulfonamides undergo photodegradation under UV-light exposure to a greater or lesser extent. The degradation products have no cytotoxic potential except sulfanilamide and have a slight impact on cell proliferation. All degradation products showed no antibacterial activity. Thus, UV-light exposure seems to represent an adequate method for inactivating sulfonamides with regard to their antimicrobial activity.

Drug Residues in Serum of Dogs Receiving Anticancer Chemotherapy

BACKGROUND The presence of drug residues in blood samples can represent an occupational hazard. However, studies on cytotoxic drug residues in serum of dogs are lacking in veterinary oncology. OBJECTIVE To evaluate possible occupational hazards associated with handling of blood samples from dogs receiving oncolytic drugs 7 days after treatment. ANIMALS Twenty-seven client-owned dogs treated for lymphoma or mast cell tumors with vincristine, vinblastine, cyclophosphamide, or doxorubicin. METHODS Prospective, observational study. Serum samples were either taken 7 days after administration of vincristine, cyclophosphamide, doxorubicin (lymphoma), and vinblastine (mast cell tumor), or 1-2 days after the last concurrent oral administration of cyclophosphamide (mast cell tumor). Additionally, serum was collected within 5 minutes of treatment. Measurement of drug residues in serum was performed by liquid chromatography tandem mass spectrometry (LC/MS/MS). RESULTS In 33 samples collected within 5 minute of treatment, the median serum concentrations were vincristine: 37 microg/L (range: 11-87 microg/L), vinblastine: 13 microg/L (range: 13-35 microg/L), cyclophosphamide: 2,484 microg/L (range: 1,209-2,778 microg/L), doxorubicin: 404 microg/L (range: 234-528 microg/L). In 81 serum samples collected 7 days after treatment vinblastine (7 microg/L) was detected in 1 sample, and cyclophosphamide (7 and 9 microg/L) in 2 samples collected 1-2 days after oral administration of cyclophosphamide. Medications were not detected in any of the other samples. CONCLUSIONS AND CLINICAL IMPORTANCE Handling of blood samples from dogs receiving oncolytic chemotherapy 7 days after treatment with vincristine, vinblastine, cyclophosphamide, and doxorubicin should not present a health hazard.

Analysis and behavior of colistin during anaerobic fermentation

A new analytical method for the determination of colistin in fermenter samples was developed followed by a study on the behavior of this substance during anaerobic fermentation. Analysis of colistin A and B was carried out by liquid chromatography-tandem mass spectrometry. Separation of the analytes was performed on a Security Guard column (4×3mm). Fourteen fermentation tests in batch as well as in continuous reactors were carried out. After 44days of anaerobic digestion of cattle manure, initially spiked with 500mg/kg of colistin sulfate, a considerable decrease of the colistin concentration to less than 1mg/kg could be observed. Furthermore, the daily production of biogas and methane was measured. A correlation between gas production and colistin concentration could not be determined. However, an increase of 10% of the cumulative methane production was observed in those fermenters spiked with an initial bolus of 500mg/kg colistin.