Rapid determination of X-ray contrast agent iomeprol in human plasma by on-line solid-phase extraction coupled with phase optimized liquid chromatography.

Phase optimized liquid chromatography (POPLC) allows for the optimized combination of column segments of any length and stationary phases with different functionalities. In this study, a simple and rapid method using POPLC coupled with on-line solid-phase extraction (SPE) for the analysis of X-ray contrast media agent iomeprol (IOM) in human plasma was developed. Because the phenyl (PH) stationary phase has strong hydrophobic and π-π interactions with IOM and iopromide (IOP, internal standard), the best separation efficiency was achieved with a 250mm×3mm homogenous PH POPLC-column. Different kinds of on-line SPE sorbents were studied, including restricted access material-alkyl diol silica (ADS), LiChrolut EN with excellent absorption capacity and hydrophilic-lipophilic-balanced Oasis HLB. The most efficient on-line sample clean-up was carried out using a fast-flow on-line purification approach with an Oasis HLB pre-column ((20mm×2mm, 30µm). This pre-column showed excellent durability and reproducibility. At least 400 samples could be analyzed with one pre-column. Each plasma sample was directly injected and analyzed within 15min. The calibration curves were linear in the range of 10-1000µg/mL. The limit of quantitation was 2.26µg/mL. The inter-day precision of this method was excellent and less than 1.44%, and the intra-day precision was less than 4.44%. The inter-day and intra-day accuracy ranged from 94.33% to 104.36% and 94.60% to 101.71%, respectively. This validated method is expected to be useful in the analysis of human plasma samples for glomerular filtration rate (GFR) measurements and assessment of kidney function.

Development and validation of an on-line multidimensional SPE-LC-MS/MS method for the quantitation of Tetrandrine in blood samples.

On-line solid-phase extraction (SPE) is becoming an increasingly widespread technique in the clean-up of complex matrices such as body fluids, prior to chromatographic analysis. The use of small SPE columns instead of disposable SPE cartridges allows multiple injections and complete automation. In addition, it decreases the cost of consumables and improves the quality of the overall analysis. Coupling of SPE with HPLC combines sample preparation and separation in one system. In this paper a validated on-line multidimensional (MD) SPE-LC-MS/MS method is described for the determination of Tetrandrine (model drug) in human blood samples. The developed method showed the applicability of direct injection of plasma samples to an on-line MD-SPE-LC-MS/MS system to determine small molecules i.e. drugs. The experimental design is unique. Quantification was through tandem mass spectrometry with positive electrospray ionization (ESI) and multiple reactions monitoring (MRM). The limit of detection was calculated as 31.98 ng/mL. The linear range of the method was between 40.0 and 800.0 ng/mL. Pharmacokinetic parameters are usually determined by analysis of drug concentrations in plasma rather than whole blood. Parameters determined using plasma data may be misleading if concentrations of drug differ between plasma and red blood cells. We successfully applied the developed method for the determination of the distribution coefficient of the model drug Tetrandrine between human red blood cells and blood plasma proteins. The determination of distribution coefficient study results demonstrated that the developed method can provide direct and accurate measurement of RBC partitioning in a model drug and could be applied for screening of other compounds for potential high RBC partition, predicting potential drug toxicity and investigating mechanisms associated with RBC partitions.

Analysis of the endogenous human serum peptides by on-line extraction with restricted-access material and HPLC-MS/MS identification.

The selective extraction of endogenous serum peptides has been a challenge due to the high abundant proteins present in serum. Here a simple on-line extraction of peptides from human serum using strong cation-exchange diol silica restricted-access materials (SCX-RAM) coupled with two-dimensional RP-RP liquid chromatography mass spectrometry was developed. The operation of the on-line extraction system is simple to use and does not need complex equipments. The two-dimensional RP-RP was proved to be orthogonal and efficient to separate peptides extracted from human serum.

Selective on-line serum peptide extraction and multidimensional separation by coupling a restricted-access material-based capillary trap column with nanoliquid chromatography-tandem mass spectrometry.

As the serum peptidome gets increasing attention for biomarker discovery, one of the important issues is how to efficiently extract the peptides from highly complex human serum for peptidome analysis. Here we developed a fully automated platform for direct injection, on-line extraction, multidimensional separation and MS detection of peptides present in human serum. A capillary SPE column packed with a novel mix mode restricted access material (RAM) exhibiting strong cation exchange and size exclusion chromatography (SCX/SEC) properties were coupled with a nanoliquid chromatography-mass spectrometry (nanoLC-MS) system. The capillary SPE column excludes the high abundant serum proteins such as HSA by size exclusion chromatography and simultaneously extracts the low molecular weight peptides by binding to sulfonic acid residues. Subsequently, the trapped peptides are eluted to a capillary LC column packed with a RP-C18 stationary phase. After injection of only 2 microL human serum to the one-dimensional nanoLC-MS system around 400 peptides could be identified. When conducting a multidimensional separation, the described SCX/SEC/RP-MS platform allows the separation and identification of 1286 peptides present in human serum by the injection and on-line processing of 20 microL human serum sample.

Extracorporeal plasma treatment for the removal of endotoxin in patients with sepsis: clinical results of a pilot study.

Despite the advances in therapeutic approaches in the management of inflammatory conditions, the incidence of sepsis is on increase in the intensive care units (ICU). In a pilot study, we investigated whether the use of an apheresis system based on DEAE-cellulose is capable of reducing the plasma concentration of endotoxin in patients with severe sepsis. We enrolled 15 intensive care patients with severe sepsis and plasma endotoxin concentrations >0.3 EU/mL. In addition to standard ICU therapy, a total of 83 apheresis treatments were performed. About 1.7 volumes of plasma (6000 mL) were treated at each apheresis session. A significant reduction in plasma endotoxin levels from a median of 0.61 to 0.39 EU/mL (-35%) could be achieved (P < 0.001). Long-term comparison of the initial and post-treatment levels after a series of five to six individual apheresis treatments also showed a statistically significant decline in circulating endotoxin, interleukin (IL)-6, C-reactive protein (CRP), fibrinogen, and an increase in cholesterol levels. Except for a transient and reversible increase of prothrombin time, no adverse events were observed in patients undergoing this new adsorption apheresis treatment. Our data show that reduction of endotoxin by extracorporeal DEAE-cellulose-based plasma treatment may prove a promising therapeutic tool for patients suffering from bacterial sepsis and proven endotoxemia.

Uptake of antineoplastic agents in pharmacy and hospital personnel. Part I: monitoring of urinary concentrations.

OBJECTIVES: There is a risk of adverse health effects for personnel with occupational exposure to antineoplastic agents. The aim of the present longitudinal study was to identify, quantify, and evaluate potential health hazards of occupationally exposed workers in pharmaceutical and oncological departments with central processing units for drug preparation. METHODS: One hundred operatives in 14 German hospital pharmacies and oncological departments underwent biological monitoring by providing urine samples up to four times over a period of 3 years. RESULTS: All antineoplastic agents that were considered (cyclophosphamide, ifosfamide, doxorubicin, epirubicin and platinum from cisplatin and carboplatin), were found in urine samples in up to 40% of participants. CONCLUSIONS: Despite standard safety precautions, such as the use of vertical laminar air flow safety cabinets, and personal protective clothing, incorporation of drugs was detected. Therefore, an environmental monitoring strategy should be developed in order to detect contamination and attempt to improve hygiene during work.

Uptake of antineoplastic agents in pharmacy personnel. Part II: study of work-related risk factors.

OBJECTIVES: This study aimed to find working conditions related to internal exposure of substances handled in centralised cytostatic drug preparation units in hospitals. Recommendations to avoid this uptake should be deduced from the results. METHOD: In a longitudinal study over 3 years, 87 pharmacy technicians and pharmacists of 14 different hospitals in Germany provided 24-h urine samples separately up to three times (three sampling cycles: cycles 1-3) at the end of a working week. Additional samples were taken after 2 days and after at least 3 weeks of absence. Cyclophosphamide and ifosfamide, doxo-, dauno- epi-, and idarubicin, and platinum deriving from cis- and carboplatin were determined in urine samples by gas chromatography/mass spectrometry, liquid chromatography (HPLC) and voltammetry. The following working conditions were assessed by questionnaire: working tasks, different ways that the workbenches were run, cleaning conditions, waste disposal, number of preparations, amount of substances handled, and use of gloves (material, thickness and changing interval). RESULTS: Two-thirds of the subjects showed at least one positive result with regard to all three cycles (56 of initially 87 subjects). Employees who only pass material that is needed for processing are affected, just as are those who only prepare administrations and those alternating in both functions (25% vs. 24.1% vs. 50.6%, respectively). The storage of waste in containers that could be opened to add waste tends to increase the risk of internal exposure of ifosfamide and cyclophosphamide (odds ratios (95% confidence interval): 0.08 (0.013-0.5) and 0.19 (0.03-1.12), respectively). The amount handled and number of preparations of cyclophosphamide for "manufacturers" were associated with internal exposure of cyclophosphamide (28.04 (1.75-448.74) and 1.22 (1.03-1.44), respectively). The total number of preparations handled by assistants seemed to increase the risk of intake of any of the substances under study [1.04 (1.00-1.08)]. CONCLUSION: Since employees who pass materials are affected in the same way as those who prepare administrations, both have to be included in reviewing protective measures. Further studies must be carried out to verify the generated hypotheses of factors related to internal exposure found in this study.