Kinetic assessment of persistent halogenated xenobiotics in cell culture models: comparison of mono- and poly-halogenated compounds.

We evaluated the suitability of single and multiple cell type cultures as model systems to characterise cellular kinetics of highly lipophilic compounds with potential ecotoxicological impact. Confluent mono-layers of human skin fibroblasts, rat astrocytoma C6 cells, non-differentiated and differentiated mouse 3T3 cells were kept in culture medium supplemented with 10% foetal calf serum. For competitive uptake experiments up to four different cell types, grown on glass sectors, were exposed for 3h to (14)C-labelled model compounds, dissolved either in organic solvents or incorporated into unilamellar lecithin liposomes. Bromo-, or chloro-benzenes, decabromodiphenylether (DBP), and dichlorodiphenyl ethylene (DDE) were tested in rather high concentration of 20 microM. Cellular toxicity was low. Compound levels were related to protein, DNA, and triglyceride contents. Cellular uptake was fast and dependent on physico-chemical properties of the compounds (lipophilicity, molecular size), formulation, and cell type. Mono-halogenated benzenes showed low and similar uptake levels (=low accumulation compounds). DBP and DDE showed much higher cellular accumulations (=high accumulation compounds) except for DBP in 3T3 cells. Uptake from liposomal formulations was mostly higher than if compounds were dissolved in organic solvents. The extent of uptake correlated with the cellular content of triglycerides, except for DBP. Uptake competition between different cell types was studied in a sectorial multi-cell culture model. For low accumulation compounds negligible differences were found among C6 cells and fibroblasts. Uptake of DDE was slightly and that of DBP highly increased in fibroblasts. Well-defined cell culture systems, especially the sectorial model, are appropriate to screen for bioaccumulation and cytotoxicity of (unknown) chemical entities in vitro.

Rapid i.v. loading with phenytoin with subsequent dose adaptation using non-steady-state serum levels and a Bayesian forecasting computer program to predict maintenance doses.

OBJECTIVE: To evaluate the suitability of a phenytoin loading dose regimen; to assess whether dose-individualization was necessary and to investigate the reliability of a Bayesian forecasting method for phenytoin dose adaptation using non-steady-state levels in hospital-admitted patients. METHOD: An initial loading dose (15 mg phenytoin acid/kg BW) was given i.v. over 4 h, followed by standardized maintenance doses given i.v. in 12-h intervals from days 1 to 5 (175 mg 70 kg BW). The evening dose of day 5 was individualized based on three serum trough levels: L1 (after 16 h), L2 (morning day 4) and L3 (morning day 5). RESULTS: Ninety of 136 consecutive patients were evaluable in a prospective study for the standardized phase; 50 of them had additional serum levels in the individualized phase. There was no exclusion of patients with interacting co-medication. Seventy-seven per cent (L1) and 68% (L3) of patients showed therapeutic values (10-20 mg/L). The prediction error of the forecasting was 3.95 mg/L, the root mean squared error 6.27 mg/L (target trough level 11 mg/L). Seventy per cent of the levels (n=50) were within the 68% confidence interval. CONCLUSION: The effectiveness and safety of the regimen with rapid i.v. loading and the necessity to individualize phenytoin dosing after day 5 were demonstrated.

Lipid peroxidation of intravenous lipid emulsions and all-in-one admixtures in total parenteral nutrition bags: the influence of trace elements.

An iodometric titration was used to assess the influence of a daily portion of trace elements on lipid peroxidation of pure lipid emulsions and lipid-containing all-in-one (AIO) admixtures by measuring the peroxide value (PV; mmol peroxides/L). A pure lipid emulsion (Intralipid 20%; Pharmacia & Upjohn, Dubendorf, Switzerland) was stored in ethylvinylacetate bags under light protection (LP) at 40 degrees C with and without trace elements. In absence of trace elements the PV of Intralipid 20% was significantly lower (day 14: 2.77 vs 18.04; p < .001). After the same time period with the same storage conditions the drop in pH was two times higher in presence of trace elements (1.54 vs 0.77). In an AIO admixture with LP stored at 2 degrees C to 8 degrees C, trace elements increased the PV from 0.04 to 0.19 mmol/L (day 29; p < .01). The drop in pH was 0.01 and 0.02 units, respectively. When stored at 20 degrees C to 30 degrees C and exposed to daylight, the PV of the AIO admixture containing trace elements reached 1.92 compared with 0.52 in their absence (day 19; p < .001) with a pH drop of 0.03 and 0.11, respectively (day 29). Although trace elements led to a much higher drop in pH in pure lipid emulsions, no obvious influence on the pH of AIO admixtures was demonstrated. To minimize lipid peroxidation, AIO admixtures should be stored light-protected and refrigerated without trace elements. The latter should be added immediately before administration or should be given separately.

Lipid peroxidation of i.v. lipid emulsions in TPN bags: the influence of tocopherols.

Four commercial i.v. lipid emulsions containing soybean oil were investigated to determine the tocopherol content. A sensitive high-performance liquid chromatography (HPLC) on a diol column was established to quantitate the tocopherol isomers in lipid emulsions. A previously described iodometric titration was used to assess the peroxide value (mmol peroxides/L). The pH was measured also. The initial tocopherol concentration ranges in three of the four commercial soybean oil-based 20% lipid emulsions studied were compared ([mg/L]: alpha: 17-23, beta: 4, gamma: 88-129, delta: 40-44). One product showed an increased alpha-tocopherol content (172 mg/L) due to supplementation during manufacture. During storage in an ethylvinyl acetate (EVA) bag at 40 degrees C under light-protection (LP) for 34 d, a lipid emulsion 20% with a natural alpha-tocopherol content showed a peroxide value (PV) of 9.18 (about 450 times the value of controls in glass bottles) with a concomittant reduction of the tocopherol isomers to 61.6% (alpha), 86.5% (gamma), and 88.9% (delta) compared to the initial values. Comparison of two lipid emulsions with different amounts of alpha-tocopherol (Lipidem 20%, B. Braun, Switzerland: 156.29 mg/L vs. Intralipid 20%, Pharmacia Upjohn, Switzerland: 8.75 mg/L) for their antioxidative capacity using the same stress conditions revealed for the emulsion with the high alpha-tocopherol content a significantly higher PV over the whole test period (after 5 wk: 33.63 vs. 6.23; P < 0.001) and an increased alpha-tocopherol decomposition (51.6% vs. 8.7%). The drop in pH was higher, also (1.9 vs. 1.0 pH units). In contrast to ordinary concentrations of about 20 mg/L, alpha-tocopherol in 20% lipid emulsions showing antioxidative properties, a supplementation with about 160 mg/L showed a prooxidative effect when exposed to ambient atmosphere in an EVA bag.

In vitro oxidation of i.v. lipid emulsions in different all-in-one admixture bags assessed by an iodometric assay and gas-liquid chromatography.

Polyunsaturated fatty acids (FAs) of intravenous (IV) lipid emulsions can peroxidize to potentially harmful lipid hydroperoxides. In order to assess in vitro peroxidation of IV fat emulsions in all-in-one (AIO) admixture bags, an iodometric titration to determine lipid hydroperoxide content expressed by the peroxide value (PV) and a gas-liquid chromatographic (GLC) assay to determine changes of the FA pattern were established. A long-chain triglyceride (LCT) and medium-chain triglyceride-LCT emulsion were compared for the PV and the pH during storage at room temperature and daylight in AIO bags made of ethylvinylacetate (EVA) and polypropylene:polyamide 7:3 (V90). In contrast to storage in glass bottles, significant peroxidation was detected in both emulsions with 0.5-3.4 mmol peroxides/L after 28 d (150 times the control PV). A pH drop of at least 0.3 (EVA) and 1.2 (V90) units was measured. Initial PVs and peroxidation kinetics of the emulsions were different; V90 material showed better barrier properties against oxygen. PV was increased by higher temperature and light exposure. The FA pattern of an LCT emulsion with a PV > 6 (storage: 40 degrees C in a dark room for 28 d in AIO bags) assayed by GLC remained unchanged. The iodometric peroxide and the GLC assay were reproducible and easy to handle. Only the iodometric method was sensitive enough to detect peroxidation effects (detection limit: 0.02 mmol peroxides/L). IV fat emulsions can be checked for lipid hydroperoxide content with the rapid iodometric assay to guarantee optimal quality of IV lipids used for AIO admixtures. To prevent peroxidation, lipids in AIO bags should be stored light-protected in a refrigerator an oxygen-tight overwrap is mandatory for extended periods.

Stability of high-dose morphine chloride injection upon heat sterilization: comparison of UV-spectroscopy and HPLC.

The stability of a preservative-free morphine chloride solution for intravenous or intrathecal use manufactured at a concentration of 40 mg/ml, near the solubility limit in water, was studied. The influence of heat and oxygen on morphine content was measured with and without autoclaving, and after additional thermal and oxidative stress. The morphine injection was stable during steam sterilization at 121 degrees C for up to 180 min if the solution was adjusted to a pH of 3.2 and if oxygen was eliminated by saturating the solution and flushing the vial with nitrogen before sealing. By adding an oxidizing agent (200 microliters H2O2 3% per 20 ml vial) before 15 min of sterilization, a decomposition of approximately 20% of morphine resulted when compared to oxygen-free control samples (P < 0.01, n = 3) High-performance liquid chromatography with UV detection (HPLC) and direct UV spectroscopy (UV) (the latter available in most hospital pharmacies for analytical purposes) were compared for specificity, precision and appropriateness for content and stability assessment of morphine solutions. UV could only be used for quantification of undecomposed morphine. Morphine degradation products of stressed solutions interfered with the direct UV assay of morphine at 286 nm, whereas these interfering components were separated by the ion-pair reversed-phase HPLC used. The results demonstrate that even in the absence of stabilizers, morphine chloride solutions may safely be sterilized for 15 min at 121 degrees C. The HPLC method was shown to be sufficiently sensitive and specific for quality control and stability assessment of morphine preparations, and, therefore, appropriate for the validation of the manufacture of morphine injection solutions in hospital pharmacies, where morphine solutions are manufactured in special strengths and volumes for individual patients' needs.

Stability of succinylcholine chloride injection.

The stability of succinylcholine chloride injection prepared by a hospital pharmacy was studied under a wide variety of conditions. Batches of succinylcholine chloride injection 10 mg/mL containing sodium chloride, methyl-4-hydroxybenzoate, hydrochloric acid, and water were prepared. Samples were tested for the effect of initial pH (3.0 and 4.2) and sterilization (steam treatment at 100 degrees C for 30 minutes and 121 degrees C for 20 minutes) on stability after three weeks; long-term stability under refrigeration (12, 17, and 23 months of storage at 4 degrees C); and the effect of storage temperature (4-6 degrees C, 20-26 degrees C, 35 degrees C, and 70 degrees C) and light exposure at various intervals up to 12 months. Samples were analyzed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Unlike heating at 121 degrees C, heating at 100 degrees C produced no significant loss of succinylcholine chloride, independent of the initial pH. Succinylcholine chloride was hydrolyzed only minimally over 23 months if the solution was stored at 4-6 degrees C. A 10% loss of drug content occurred if solutions were kept at 20-26 degrees C for five months, at 35 degrees C for one month, or at 70 degrees C for one day. Initial degradation was slowed if the solution was protected from light. The assessments by TLC proved to be more sensitive than the HPLC measurements. Succinylcholine chloride injection sterilized at 100 degrees C for 30 minutes can be stored for up to five months at room temperature if protected from light. The preparation is stable for at least two years under refrigeration.