RESUMO
The efficacy of DNA-damaging anticancer drugs is highly influenced by cellular DNA repair capacity, and by inhibiting the relevant DNA repair pathway, efficacy of alkylating agents may be increased. Therefore, combining DNA repair inhibitors with anticancer agents that selectively target tumor tissue should improve cancer treatment. The objective of this study was to test the hypothesis that cotreatment of cancer cells with acylfulvene (AF, alkylating agent) and UCN-01 (DNA repair inhibitor) would improve drug efficacy and promote the persistence of DNA adducts. Previous data regarding the relative susceptibility of repair proficient versus deficient cells toward an AF analogue suggests that corresponding adducts are repaired by nuclear excision repair (NER), a cellular process that has been shown to be prevented with UCN-01. In this study, cells were cotreated with nontoxic levels of UCN-01 together with increasing doses of AF. The efficacy of AF was assessed by measuring cytotoxicity and DNA adducts. In addition, cells were cotreated with nontoxic levels of methoxyamine, a known base excision repair (BER) inhibitor, to determine if inhibiting BER also promotes cytotoxicity of AF. DNA-adducts were measured in a sensitive and precise manner by using stable isotope-labeled mass spectrometry analysis. The data obtained in this study demonstrate for the first time that pharmacological inhibition of the NER pathway of DNA repair leads to the persistence of AF-specific adducts and promotes AF cytotoxicity.
Assuntos
Antineoplásicos Alquilantes/toxicidade , Reparo do DNA/efeitos dos fármacos , Sesquiterpenos/toxicidade , Compostos de Espiro/toxicidade , Estaurosporina/análogos & derivados , Antineoplásicos Alquilantes/química , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Adutos de DNA/análise , Adutos de DNA/metabolismo , Enzimas Reparadoras do DNA/antagonistas & inibidores , Enzimas Reparadoras do DNA/metabolismo , Sinergismo Farmacológico , Células HT29 , Humanos , Sesquiterpenos/química , Espectrometria de Massas por Ionização por Electrospray , Compostos de Espiro/química , Estaurosporina/química , Estaurosporina/toxicidadeRESUMO
Illudin S and its semisynthetic analogue acylfulvene (AF) are structurally similar but elicit different biological responses. AF is a bioreductive alkylating anticancer agent with a favorable therapeutic index, while illudin S is in general highly toxic. AF toxicity is dependent on the reductase enzyme prostaglandin reductase 1 (PTGR1) for activation to a cytotoxic reactive intermediate. While illudin S can be metabolized by PTGR1, available data suggest that its toxicity does not correspond with PTGR1 function. The goal of this study was to understand how drug cytotoxicity relates to cellular bioactivation capacity and the identity and quantity of AF- or illudin S-DNA adducts. The strategy involved identification of novel illudin S-DNA adducts and their quantitation in a newly engineered SW-480 colon cancer cell line that stably overexpresses PTGR1 (PTGR1-480). These data were compared with cytotoxicity data for both compounds in PTGR1-480 versus normal SW-480 cells, demonstrating that AF forms more DNA adducts and is more cytotoxic in cells with higher levels of PTGR1, whereas illudin S cytotoxicity and adduct formation are not influenced by PTGR1 levels. Results are discussed in the context of an overall model for how changes in relative propensities of these compounds to undergo cellular processes, such as bioactivation, contributes to DNA damage, and cytotoxicity.
Assuntos
Antibióticos Antineoplásicos/química , Cromatografia Líquida de Alta Pressão , Adutos de DNA/análise , Sesquiterpenos/química , Compostos de Espiro/química , Alquilação , Animais , Antibióticos Antineoplásicos/metabolismo , Antibióticos Antineoplásicos/toxicidade , Bovinos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , DNA/química , Humanos , Sesquiterpenos Policíclicos , Sesquiterpenos/metabolismo , Sesquiterpenos/toxicidade , Espectrometria de Massas por Ionização por Electrospray , Compostos de Espiro/metabolismoRESUMO
Microfluidic technology is providing new routes toward advanced cell and tissue culture models to better understand human biology and disease. Many advanced devices have been made from poly(dimethylsiloxane) (PDMS) to enable experiments, for example, to study drug metabolism by use of precision-cut liver slices, that are not possible with conventional systems. However, PDMS, a silicone rubber material, is very hydrophobic and tends to exhibit significant adsorption and absorption of hydrophobic drugs and their metabolites. Although glass could be used as an alternative, thermoplastics are better from a cost and fabrication perspective. Thermoplastic polymers (plastics) allow easy surface treatment and are generally transparent and biocompatible. This study focuses on the fabrication of biocompatible microfluidic devices with low adsorption properties from the thermoplastics poly(methyl methacrylate) (PMMA), polystyrene (PS), polycarbonate (PC), and cyclic olefin copolymer (COC) as alternatives for PDMS devices. Thermoplastic surfaces were oxidized using UV-generated ozone or oxygen plasma to reduce adsorption of hydrophobic compounds. Surface hydrophilicity was assessed over 4 weeks by measuring the contact angle of water on the surface. The adsorption of 7-ethoxycoumarin, testosterone, and their metabolites was also determined after UV-ozone treatment. Biocompatibility was assessed by culturing human hepatoma (HepG2) cells on treated surfaces. Comparison of the adsorption properties and biocompatibility of devices in different plastics revealed that only UV-ozone-treated PC and COC devices satisfied both criteria. This paper lays an important foundation that will help researchers make informed decisions with respect to the materials they select for microfluidic cell-based culture experiments.
Assuntos
Materiais Biocompatíveis/metabolismo , Cicloparafinas/metabolismo , Técnicas Analíticas Microfluídicas/instrumentação , Cimento de Policarboxilato/metabolismo , Polimetil Metacrilato/metabolismo , Poliestirenos/metabolismo , Técnicas de Cultura de Tecidos/instrumentação , Adsorção , Materiais Biocompatíveis/química , Sobrevivência Celular , Cicloparafinas/química , Desenho de Equipamento , Células Hep G2 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Oxirredução , Cimento de Policarboxilato/química , Polimetil Metacrilato/química , Poliestirenos/químicaRESUMO
Successful vitrification of organ slices is hampered by both osmotic stress and chemical toxicity of cryoprotective agents (CPAs). In the present study, we focused on the effect of osmotic stress on the viability of precision-cut liver slices (PCLS) by comparing different CPA solutions and different methods of loading and unloading the slices with the CPAs. For this purpose, we developed a gradient method to load and unload CPAs with the intention of minimizing sudden changes in osmolarity and thereby avoiding osmotic stress in the slices in comparison with the commonly used step-wise loading/unloading approach. With this gradient method, the CPA solution was introduced at a constant rate into a specially designed mixing chamber containing the slices. We showed that immediate mixing of the infused CPA and the chamber constituents occurred, which enabled us to control the CPA concentration to which PCLS were exposed as a function of time. With this method, CPA concentration versus time profiles were varied using various commercially available CPA mixtures [VMP, VM3, M22, and modified M22 (mM22)]. The viability of PCLS was determined after CPA loading and unloading and subsequent incubation during 3h at 37°C. Despite the reduction of osmotic stress, the viability of slices did not improve with gradual loading and unloading and remained considerably lower than that of untreated slices. The toxicity of the three CPA solutions did not correlate with either their potential osmotic effects or their total concentrations, and did not change strongly with exposure time in 100% CPA. The most likely explanation for these observations is that PCLS are not very sensitive to osmotic changes of the magnitude imposed in our study, and chemical toxicity of the CPA solutions is the main barrier to be overcome. The chemical toxicity of the CPAs used in this study probably originates from a source other than the total concentration of the solutions. The presented gradient method using the specially designed chamber is more time and cost effective than the step-wise method and can be universally applied to efficiently evaluate different CPA solutions.
Assuntos
Crioprotetores/metabolismo , Fígado/metabolismo , Soluções para Preservação de Órgãos/metabolismo , Preservação de Órgãos/instrumentação , Trifosfato de Adenosina/metabolismo , Animais , Desenho de Equipamento , Fígado/ultraestrutura , Masculino , Preservação de Órgãos/métodos , Osmose , Pressão Osmótica , Permeabilidade , Ratos , Ratos Wistar , VitrificaçãoRESUMO
A novel approach for on-line monitoring of drug metabolism in continuously perifused, precision-cut liver slices (PCLS) in a microfluidic system has been developed using high-performance liquid chromatography with UV detection (HPLC-UV). In this approach, PCLS are incubated in a microfluidic device made of poly(dimethylsiloxane) (PDMS) by continuous, single-pass perifusion with fresh medium. Two syringe pumps are incorporated into the system to infuse substrates or inhibitors at varying concentrations into the perfusion medium just before the chip entrance. The medium containing the metabolites produced by the PCLS is directed toward an injection loop. Once filled, the content of this injection loop is automatically injected onto an HPLC for analysis. The on-line analysis of metabolites was tested by using the substrate, 7-hydroxycoumarin (7-HC). Rapid switching between substrate and solvent control was possible, and a direct metabolic response of the liver slice to perifusion with substrate was detected. Very stable phase II metabolism over a period of 24 h was observed. The inhibitory effect of phloxine B on the formation of 7-hydroxycoumarin glucuronide (phase II product of 7-HC) was also investigated. Phloxine B was injected into the incubation medium in increasing concentrations varying from 0 to 200 µM. The results showed a concentration-dependent inhibition of 7-HC glucuronide formation and allowed the calculation of an IC50 value (concentration in which 50% of the enzyme is inhibited) of â¼85 µM using one single liver slice. On-line detection was also shown to be advantageous for the detection of unstable metabolites. This was demonstrated by determination of the metabolites of the drug diclofenac. The reactive metabolite, acyl glucuronide, was detected at relatively high concentrations which remained very constant over a period of 4 h. In contrast, only low and decreasing amounts of diclofenac acyl glucuronide could be measured in the conventional well-plate incubation system. The advantages of this novel on-line analysis system for PCLS include the capability to obtain direct information about tissue function, assess the concentration dependence of drug-drug interactions in one single slice, and detect unstable metabolites. The system also enables fast analysis without the need to store samples, thus eliminating the associated freeze-thaw problems, and allows the simultaneous analysis of multiple metabolites.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Sistemas On-Line , Preparações Farmacêuticas/metabolismo , Animais , Diclofenaco/metabolismo , Azul de Eosina I/farmacologia , Técnicas In Vitro , Inativação Metabólica , Fígado/citologia , Masculino , Microtomia , Ratos , Ratos Wistar , Umbeliferonas/metabolismoRESUMO
A microfluidic-based biochip made of poly-(dimethylsiloxane) was recently reported for the first time by us for the incubation of precision-cut liver slices (PCLS). In this system, PCLS are continuously exposed to flow, to keep the incubation environment stable over time. Slice behavior in the biochip was compared with that of slices incubated in well plates, and verified for 24 h. The goal of the present study was to extend this incubation time. The viability and metabolic activity of precision-cut rat liver slices cultured in our novel microflow system was examined for 72 h. Slices were incubated for 1, 24, 48, and 72 h, and tested for viability (enzyme leakage (lactate dehydrogenase)) and metabolic activity (7-hydroxycoumarin (phase II) and 7-ethoxycoumarin (phase I and II)). Results show that liver slices retained a higher viability in the biochip when embedded in a hydrogel (Matrigel) over 72 h. This embedding prevented the slices from attaching to the upper polycarbonate surface in the microchamber, which occurred during prolonged (>24 h) incubation in the absence of hydrogel. Phase II metabolism was completely retained in hydrogel-embedded slices when medium supplemented with dexamethasone, insulin, and calf serum was used. However, phase I metabolism was significantly decreased with respect to the initial values in gel-embedded slices with medium supplements. Slices were still able to produce phase I metabolites after 72 h, but at only about â¼10% of the initial value. The same decrease in metabolic rate was observed in slices incubated in well plates, indicating that this decrease is due to the slices and medium rather than the incubation system. In conclusion, the biochip model was significantly improved by embedding slices in Matrigel and using proper medium supplements. This is important for in vitro testing of drug metabolism, drug-drug interactions, and (chronic) toxicity.
Assuntos
Fígado/metabolismo , Preparações Farmacêuticas/metabolismo , Análise Serial de Tecidos/instrumentação , Animais , Desenho de Equipamento , Hidrogel de Polietilenoglicol-Dimetacrilato/metabolismo , Masculino , Ratos , Ratos WistarRESUMO
There is an increasing interest in controlled release systems for local therapy in the treatment of human and equine joint diseases, aiming for optimal intra-articular concentrations with no systemic side effects. In this study, the intra-articular tolerability and suitability for local and sustained release of tacrolimus (FK506) from monospheres composed of [PDLA-PEG1000]-b-PLLA multiblock copolymers were investigated. Unloaded and tacrolimus-loaded (18.4 mg tacrolimus/joint) monospheres were injected into the joints of six healthy horses, with saline and hyaluronic acid (HA) in the contralateral joints as controls. Blood and synovial fluid were analysed for the tacrolimus concentration and biomarkers for inflammation and cartilage metabolism. After an initial burst release, sustained intra-articular tacrolimus concentrations (>20 ng/mL) were observed during the 42 days follow-up. Whole-blood tacrolimus levels were below the detectable level (<0.5 ng/mL). A transient inflammatory reaction was observed for all substances, evidenced by increases of the synovial fluid white blood cell count and total protein. Prostaglandin and glycosaminoglycan release were increased in joints injected with unloaded monospheres, which was mitigated by tacrolimus. Both tacrolimus-loaded monospheres and HA transiently increased the concentration of collagen II cleavage products (C2C). A histologic evaluation of the joints at the endpoint showed no pathological changes in any of the conditions. Together, these results indicate the good biocompatibility of intra-articular applied tacrolimus-loaded monospheres combined with prolonged local drug release while minimising the risk of systemic side effects. Further evaluation in a clinical setting is needed to determine if tacrolimus-loaded monospheres can be beneficial in the treatment of inflammatory joint diseases in humans and animals.
RESUMO
Over the past two decades, it has become increasingly clear that the intestine, in addition to the liver, plays an important role in the metabolism of xenobiotics. Previously, we developed a microfluidic-based in vitro system for the perifusion of precision-cut liver slices for metabolism studies. In the present study, the applicability of this system for the perifusion of precision-cut intestinal slices, and for the sequential perifusion of intestinal and liver slices, all from rat, was tested to mimic the in vivo first pass situation. Intestinal and liver slices, exposed to the substrates 7-ethoxycoumarin (7-EC), 7-hydroxycoumarin (7-HC) and lidocaine (Li), exhibited similar metabolic rates in the biochip and in the well plates for periods of at least 3 h. The metabolic rate remained the same when two slices were placed in adjacent microchambers and perifused sequentially. In addition, the system has been adapted to sequentially perifuse intestinal and liver tissue slices in a two-compartment co-culture perfusion system with a continuous flow of medium. It becomes possible to direct metabolites or other excreted compounds formed by an intestinal slice in the first compartment to the second compartment containing a liver slice. The intestine does not influence liver metabolism for these substrates. The interplay between these two organs was demonstrated by exposing the slices to the primary bile acid, chenodeoxycholic acid (CDCA). CDCA induced the expression of fibroblast growth factor 15 (FGF15) in the intestinal slice, which resulted in a stronger down-regulation of the enzyme, cytochrome P450 7A1 (CYP7A1), in the liver slice in the second compartment than when the liver slice was exposed to CDCA in a single-microchamber biochip. We thus demonstrate in this paper that intestinal slices, in addition to liver slices, remain functional in the biochip under flow conditions, and that the two-microchamber biochip has great potential for the study of interorgan effects. This is the first example of the incorporation of both liver and intestinal slices in a microfluidic device. Use of this microfluidic system will improve our insight into interorgan interactions and elucidate as yet unknown mechanisms involved in toxicity, gene regulation and drug-drug interactions.
Assuntos
Cumarínicos/farmacocinética , Análise de Injeção de Fluxo/instrumentação , Mucosa Intestinal/metabolismo , Lidocaína/farmacocinética , Fígado/metabolismo , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas de Cultura de Órgãos/instrumentação , Animais , Cumarínicos/administração & dosagem , Desenho de Equipamento , Análise de Falha de Equipamento , Lidocaína/administração & dosagem , Masculino , Ratos , Ratos WistarRESUMO
Early detection of kinetic, metabolic, and toxicity (ADME-Tox) profiles for new drug candidates is of crucial importance during drug development. This article describes a novel in vitro system for the incubation of precision-cut liver slices (PCLS) under flow conditions, based on a poly(dimethylsiloxane) (PDMS) device containing 25-microL microchambers for integration of the slices. The microdevice is coupled to a perifusion system, which enables a constant delivery of nutrients and oxygen and a continuous removal of waste products. Both a highly controlled incubation environment and high metabolite detection sensitivity could be achieved using microfluidics. Liver slices were viable for at least 24 h in the microdevice. The compound, 7-ethoxycoumarin (7-EC), was chosen to test metabolism, since its metabolism includes both phase I and phase II metabolism and when tested in the conventional well plate system, correlates well with the in vivo situation (De Kanter et al. 2004. Xenobiotica 34(3): 229-241.). The metabolic rate of 7-EC was found to be 214 +/- 5 pmol/min/mg protein in the microdevice, comparable to well plates, and was constant over time for at least 3 h. This perifusion system better mimics the in vivo situation, and has the potential to significantly contribute to drug metabolism and toxicology studies of novel chemical entities.
Assuntos
Cumarínicos/toxicidade , Inativação Metabólica , Fígado/metabolismo , Microfluídica , Toxicologia/instrumentação , Animais , Sobrevivência Celular , Cumarínicos/química , Concentração de Íons de Hidrogênio , Masculino , Microfluídica/economia , Estrutura Molecular , Ratos , Ratos Wistar , Técnicas de Cultura de Tecidos , Toxicologia/métodosRESUMO
Injectable thermogels based on poly(ε-caprolactone-co-lactide)-b-poly(ethylene glycol)-b-poly(ε-caprolactone-co-lactide) (PCLA-PEG-PCLA) containing an acetyl- or propyl endcap and loaded with celecoxib were developed for local drug release. The aim of this study was to determine the effects of the composition of the celecoxib/PCLA-PEG-PCLA formulation on their in vivo drug release characteristics. Furthermore, we want to obtain insight into the in vitro-in vivo correlation. Different formulations were injected subcutaneously in rats and blood samples were taken for a period of 8â¯weeks. Celecoxib half-life in blood increased from 5â¯h for the bolus injection of celecoxib to more than 10â¯days for the slowest releasing gel formulation. Sustained release of celecoxib was obtained for at least 8â¯weeks after subcutaneous administration. The release period was prolonged from 3 to 6-8â¯weeks by increasing the injected volume from 100 to 500⯵L, which also led to higher serum concentrations in time. Propyl endcapping of the polymer also led to a prolonged release compared to the acetyl endcapped polymer (49 versus 21â¯days) and at equal injected dose of the drug in lower serum concentrations. Increasing the celecoxib loading from 10â¯mg/mL to 50â¯mg/mL surprisingly led to prolonged release (28 versus 56â¯days) as well as higher serum concentrations per time point, even when corrected for the higher dose applied. The in vivo release was about twice as fast compared to the in vitro release for all formulations. Imaging of organs of mice, harvested 15â¯weeks after subcutaneous injection with polymer solution loaded with infrared-780 labelled dye showed no accumulation in any of these harvested organs except for traces in the kidneys, indicating renal clearance. Due to its simplicity and versatility, this drug delivery system has great potential for designing an injectable to locally treat osteoarthritis, and to enable tuning the gel to meet patient-specific needs.
Assuntos
Celecoxib/administração & dosagem , Celecoxib/farmacocinética , Inibidores de Ciclo-Oxigenase 2/administração & dosagem , Inibidores de Ciclo-Oxigenase 2/farmacocinética , Poliésteres/química , Polietilenoglicóis/química , Animais , Disponibilidade Biológica , Celecoxib/química , Inibidores de Ciclo-Oxigenase 2/química , Preparações de Ação Retardada , Composição de Medicamentos , Sistemas de Liberação de Medicamentos , Excipientes , Géis , Meia-Vida , Hidrogéis , Injeções Subcutâneas , Masculino , Camundongos , Ratos , Ratos WistarRESUMO
Microfluidic technologies enable the fabrication of advanced in vitro systems incorporating liver tissue or cells to perform metabolism and toxicity studies for drugs and other xenobiotics. The use of microfluidics provides the possibility to utilize a flow of medium, thereby creating a well-controlled microenvironment. The general goals of most in vitro systems in drug research are to optimally mimic the in vivo situation, and to minimize the number of animals required for preclinical studies. Moreover, they may contribute to a reduced attrition rate of drugs at a late stage of the drug development process; this is especially true if human tissue or cells are used. A number of factors are important in achieving good in vivo predictability in microfluidic systems, of which the biological system itself (cells or tissue) and the incubation conditions are the most important. The last couple of years have seen various microfluidic-based in vitro systems being developed to incorporate many different cells and/or tissues. In this review, microfluidics-based in vitro systems realized to study liver metabolism and toxicity are summarized and discussed with respect to their applications, advantages, and limitations. The biological basis of these systems is evaluated, and incubation conditions considered. Precise control of the cell or tissue microenvironment is a key advantage of using microfluidic technologies, and the benefits of exposing the cells to medium flow are demonstrated. Special attention is also paid to the incorporation of multiple cell types or tissues into a microfluidic device for the investigation of interorgan interactions, which are difficult if not impossible to study in conventional systems.
Assuntos
Técnicas de Cultura de Células/instrumentação , Análise de Injeção de Fluxo/instrumentação , Fígado/efeitos dos fármacos , Fígado/metabolismo , Técnicas Analíticas Microfluídicas/instrumentação , Farmacocinética , Testes de Toxicidade/instrumentação , Animais , Bioensaio/instrumentação , Desenho de Equipamento , Humanos , Fígado/patologiaRESUMO
Early information on the metabolism and toxicity properties of new drug candidates is crucial for selecting the right candidates for further development. Preclinical trials rely on cell-based in vitro tests and animal studies to characterize the in vivo behavior of drug candidates, although neither are ideal predictors of drug behavior in humans. Improving in vitro systems for preclinical studies both from a technological and biological model standpoint thus remains a major challenge. This article describes how microfluidics can be exploited to come closer to this goal in combination with precision-cut liver slices (PCLS) as an improved organomimetic system. Recently, we developed a novel microfluidic-based system incorporating a microchamber for slice perifusion to perform drug metabolism studies with mammalian PCLS under continuous flow. In the present study, the viability and metabolism of human PCLS were assessed by the measurement of the leakage of liver-specific enzymes and metabolism of four different substrates: lidocaine, 7-hydroxycoumarin, 7-ethoxycoumarin, and testosterone. All experiments were verified with well plates, an excellent benchmark for these experiments. Clearly, however, human tissue is not readily available, and it is worth considering how to perform a maximum number of informative experiments with small amounts of material. In one approach, the microfluidic system was coupled to an HPLC system to allow on-line monitoring and immediate detection of unstable metabolites, something that is generally not possible with conventional well-plate systems. This novel microfluidic system also enables the in vitro measurement of interorgan interactions by connecting microchambers containing different organ slices in series for sequential perfusion. This versatile experimental system has the potential to yield more information about the metabolic profiles of new drug candidates in human and animal tissues in an early stage of development compared with well plates alone.
Assuntos
Citometria de Fluxo , Fígado/metabolismo , Automação Laboratorial , Cromatografia Líquida de Alta Pressão , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Fígado/patologia , Coloração e RotulagemRESUMO
Poor repeatability of peak areas is a problem frequently encountered in peptide analysis with nanoLiquid Chromatography coupled on-line with Mass Spectrometry (nanoLC-MS). As a result, quantitative analysis will be seriously hampered unless the observed variability can be corrected in some way. Currently, labeling techniques or addition of internal standards are often applied for this purpose. However, these procedures are elaborate and error-prone and may render complex samples even more complex. Moreover, whenever poor repeatability results from variable recovery, not just quantification, but also sensitivity is affected. We have studied the parameters influencing the repeatability of chromatographic peak areas for a model set of proteolytic peptides (i.e., a cytochrome c tryptic digest) in nanoLC-MS analysis. It is demonstrated that repeatability issues are mainly due to poor recovery of peptides from the sample vial. Problems are largely resolved by addition of an organic modifier to the sample vial to improve solubility of the peptides, but care needs to be taken not to lose peptides due to reduced affinity for reversed-phase materials. Good results are obtained when applying dimethylsulfoxide (DMSO) for this purpose. When applying DMSO, repeatability increases, and the limit of detection (LOD) decreases. For the most hydrophobic peptides, a gain in LOD of at least an order of magnitude is obtained. In an aqueous sample containing 0.1% formic acid (FA), it is possible to detect 100-200 fmol of peptide, whereas +/-10 fmol can be detected in a sample containing 5% FA and 25% DMSO (10 microL injections).