Small Volumes Of Biological Samples Research Articles

Pharmacokinetic analysis of trichloroethylene metabolism in male B6C3F1 mice: Formation and disposition of trichloroacetic acid, dichloroacetic acid, S-(1,2-dichlorovinyl)glutathione and S-(1,2-dichlorovinyl)- l-cysteine

Trichloroethylene (TCE) is a well-known carcinogen in rodents and concerns exist regarding its potential carcinogenicity in humans. Oxidative metabolites of TCE, such as dichloroacetic acid (DCA) and trichloroacetic acid (TCA), are thought to be hepatotoxic and carcinogenic in mice. The reactive products of glutathione conjugation, such as S-(1,2-dichlorovinyl)- l-cysteine (DCVC), and S-(1,2-dichlorovinyl) glutathione (DCVG), are associated with renal toxicity in rats. Recently, we developed a new analytical method for simultaneous assessment of these TCE metabolites in small-volume biological samples. Since important gaps remain in our understanding of the pharmacokinetics of TCE and its metabolites, we studied a time-course of DCA, TCA, DCVG and DCVG formation and elimination after a single oral dose of 2100 mg/kg TCE in male B6C3F1 mice. Based on systemic concentration-time data, we constructed multi-compartment models to explore the kinetic properties of the formation and disposition of TCE metabolites, as well as the source of DCA formation. We conclude that TCE-oxide is the most likely source of DCA. According to the best-fit model, bioavailability of oral TCE was ∼ 74%, and the half-life and clearance of each metabolite in the mouse were as follows: DCA: 0.6 h, 0.081 ml/h; TCA: 12 h, 3.80 ml/h; DCVG: 1.4 h, 16.8 ml/h; DCVC: 1.2 h, 176 ml/h. In B6C3F1 mice, oxidative metabolites are formed in much greater quantities (∼ 3600 fold difference) than glutathione-conjugative metabolites. In addition, DCA is produced to a very limited extent relative to TCA, while most of DCVG is converted into DCVC. These pharmacokinetic studies provide insight into the kinetic properties of four key biomarkers of TCE toxicity in the mouse, representing novel information that can be used in risk assessment.

Quantitative measurements of cell-cell signaling peptides with single-cell MALDI MS.

Cell-to-cell signaling peptides play important roles in neurotransmission, neuromodulation, and hormonal signaling. Significant progress has been achieved in qualitative investigations of signaling peptides in the nervous system using single cell matrix-assisted laser desorption/ionization mass spectrometry. However, quantitative information about signaling peptides is difficult to obtain with this approach because only small amounts of analytes are available for analysis. Here we describe several methods for quantitative microanalysis of peptides in individual Aplysia californica neurons and small pieces of tissue. Stable isotope labeling with d0- and d4-succinic anhydride and iTRAQ reagents has been successfully adopted for relative quantitation of nanoliter volume samples containing the Aplysia insulin Cβ peptide. Comparative analysis of the Cβ peptide release site, the upper labial nerve, and its synthesis location, the F- and C-clusters, shows that the release site possesses almost three times more of this compound. The method of standard addition permits absolute quantitation of the physiologically active neuropeptide cerebrin from small structures, including nerves and neuronal clusters, in the femtomole range with a limit of detection of 19 fmol. The simplicity of these methods and the commercial availability of the reagents allow quantitative measurements from a variety of small-volume biological samples.

Use of the 1‐mm micro‐probe for metabolic analysis on small volume biological samples

Endogenous metabolites are promising diagnostic end-points in cancer research. Clinical application of high-resolution NMR spectroscopy is often limited by extremely low volumes of human specimens. In the present study, the use of the Bruker 1-mm high-resolution TXI micro-probe was evaluated in the elucidation of metabolic profiles for three different clinical applications with limited sample sizes (body fluids, isolated cells and tissue biopsies). Sample preparation and (1)H-NMR metabolite quantification protocols were optimized for following oncology-oriented applications: (i) to validate the absolute concentrations of citrate and spermine in human expressed prostatic specimens (EPS volumes 5 to 10 microl: prostate cancer application); (ii) to establish the metabolic profile of isolated human lymphocytes (total cell count 4 x 10(6): chronic myelogenous leukaemia application); (iii) to assess the metabolic composition of human head-and-neck cancers from mouse xenografts (biopsy weights 20 to 70 mg: anti-cancer treatment application). In this study, the use of the Bruker 1-mm micro-probe provides a convenient way to measure and quantify endogenous metabolic profiles of samples with a very low volume/weight/cell count.

Determination of Volatile Organic Compounds in Biological Samples Using Headspace Solid-Phase Microextraction and Gas Chromatography: Toluene and Styrene

Epidemiological and laboratory investigations have shown that toluene and styrene are toxic compounds that lead to impairment of the nervous system. To quantitate toluene and styrene in biological samples, liquid-liquid phase, headspace (HS), and solid-phase microextraction (SPME) methods are generally used. Most of these methods are not sensitive enough for applications involving small sample volumes. Here, we present a method for quantitative analysis of low concentrations of styrene and toluene in very small volumes of biological samples using HS-SPME and gas chromatography (GC) equipped with a flame-ionization detector. The method was developed by optimizing operating parameters that affect the HS-SPME-GC process [i.e., desorption time (30 s), depth of the fiber in the GC injection port (3.7 cm), adsorption time (4 min), and adsorption temperature (room temperature)]. It has a wide range of linearity (0.5-500 ng/10 microL), high precision (coefficient of variation < 5%), good accuracy (deviation < 11%), and low detection limits of 0.13 and 0.08 ng/10 microL for styrene and toluene in serum, respectively. This analytical technique can be applied to the estimation of styrene and toluene in small volumes of biological fluids (blood, serum, and perilymph) and tissues of low lipid content (cochlea).

Recent applications on isotope ratio measurements by ICP-MS and LA-ICP-MS on biological samples and single particles

Inductively coupled plasma mass spectrometry (ICP-MS) and laser ablation ICP-MS (LA-ICP-MS) have proved themselves to be powerful and sensitive inorganic mass spectrometric techniques for analysing stable and radioactive isotopes in different application fields because of their high sensitivity, low detection limits, good accuracy and precision. New applications of ICP-MS focus on tracer experiments and the development of isotope dilution techniques together with nanoflow injections for the analysis of small volumes of biological samples. Today, LA-ICP-MS is the method of choice for direct determination of metals, e.g., on protein bands in gels after the gel electrophoresis of protein mixtures. Tracer experiments using highly enriched 65Cu were utilized in order to study the formation of metal-binding bovine serum proteins. A challenging task for LA-ICP-MS is its application as an imaging mass spectrometric technique for the production of isotope images (e.g., from thin sections of brain tissues stained with neodymium). In this paper, we demonstrate the application of imaging mass spectrometry on single particles (zircon and uranium oxide). Single Precambrian zircon crystals from the Baltic Shield were investigated with respect to isotope ratios using LA-ICP-MS for age dating. The U–Pb age was determined from the isochrone with (1.48 ± 0.14) × 10 9 a. Using isotope ratio measurements on 10 nuclear uranium oxide single particles the 235U/ 238U isotope ratio was determined to be 0.032 ± 0.004. This paper describes recent developments and applications of isotope ratio measurements by ICP-MS and LA-ICP-MS on biological samples and single particles.

Simultaneous determination of anions in nanoliter volumes

The study of renal tubular transport requires the ability to accurately measure ion concentrations in samples taken from single tubules. Sample collection and analysis are laborious, so methods allowing determination of multiple ion species in a small volume sample are advantageous. This article describes a method for the simultaneous analysis of anions at physiologic concentrations in nanoliter volumes of tubular fluid. The analysis is performed using capillary zone electrophoresis. Diluted samples are moved along a capillary by bulk transport and separated according to charge and size. Peaks corresponding to anions are obtained by ultraviolet (UV) detection; peak area is proportional to ion concentration. The anions chloride, nitrate, citrate, phosphate, and bicarbonate were separated in less than 4 minutes, and iothalamate in less than 5 minutes. Simultaneous quantitative analysis was performed for chloride, phosphate, and bicarbonate, demonstrating detection limits of 12 fmol for chloride, 12 fmol for phosphate, and 72 fmol for bicarbonate. A comparison between this method and a flow-through microfluorimeter analysis of chloride showed good agreement between the two micro-methods. Illustrative data from proximal and distal tubular fluid samples obtained by micropuncture (volume 30-70 nL) are given, as are results from urine samples. Results for chloride, phosphate, and bicarbonate in control material are in close agreement with the certified values, while values in tubular fluid are in accordance with previously published results. This method provides a straightforward means of analyzing multiple anions in small volume biological samples.

Liquid-phase microextraction based on carrier mediated transport combined with liquid chromatography–mass spectrometry: New concept for the determination of polar drugs in a single drop of human plasma

Recently, we demonstrated for the first time liquid-phase microextraction (LPME) of polar drugs based on carrier mediated transport. In this new extraction technique, selected analytes were extracted as ion-pairs from small volumes of biological samples, through a thin layer of a water immiscible organic solvent immobilised in the pores of a porous hollow fibre (liquid membrane), and into a μl volume of an acidic aqueous acceptor solution placed inside the lumen of the hollow fibre. In the current paper, this new extraction technique was combined with liquid chromatography–mass spectrometry (LC–MS) for the first time. Carrier mediated LPME was evaluated for several new model drugs (0.01 < log P < 1.76), the sample clean-up aspects were investigated in detail, and this new extraction technique was fully validated for the first time. Extractions were performed from 50 μl of human plasma samples, which provided sufficient material in combination with LC–MS. Sodium octanoate (50 mM) was added to the sample as carrier, 1-octanol (≈15 μl) was used as the liquid membrane in the wall of the hollow fibre, and 50 mM HCl was utilized as acceptor solution in the lumen of the hollow fibre. The addition of carrier to the samples was found to significantly improve extraction recoveries for the polar drugs tested, providing recoveries in the range 16–78%. Validation was accomplished for atenolol and cimetidine. Limits of quantification (S/N = 5) from 50 μl of plasma were 25 and 50 ng/ml for atenolol and cimetidine, respectively. The intra-day precision (R.S.D.) ranged from 7.8 to 17.2% and from 9.5 to 14.1% for atenolol and cimetidine, respectively, and corresponding inter-day precisions (R.S.D.) were within 6.7–1.4% and 7.7–20.3%. The method was linear in the range 25–1500 ng/ml for atenolol ( r = 0.992), and 50–3500 ng/ml for cimetidine ( r = 0.976). The accuracy of the method was found to be in range 89.1–99.6% and 83.4–86% for atenolol and cimetidine, respectively. The sample clean-up obtained by carrier mediated LPME was excellent, providing a significantly lower back-ground level in total ion current chromatograms by LC–MS as compared to protein precipitation.

Spurious serotonin dimer formation using electrokinetic injection in capillary electrophoresis from small volume biological samples

One normally assumes that the analytical measurement process does not introduce spurious compounds. Capillary electrophoresis is a separation method frequently used for small-volume biological measurements. We demonstrate the potential for creating new peaks in a capillary electropherogram when using electrokinetic injections and illustrate the potential deleterious effects with biological samples involving serotonin and nitric oxide measurements. Specifically, when measuring the serotonin content from individual neurons using electrokinetic injections from 360 nL stainless steel vials, we detect a new peak that we identify as a serotonin dimer. We do not observe this peak when using hydrodynamic injections.

Quantification of Doxorubicin and metabolites in rat plasma and small volume tissue samples by liquid chromatography/electrospray tandem mass spectroscopy

The anthracycline Doxorubicin (DXR) is used widely for the treatment of human malignancies, and drug delivery technologies are under investigation to enhance antitumor selectivity and effectiveness. A liquid chromatography–tandem mass spectroscopy (LC–MS/MS) method was developed to identify and quantify DXR and key metabolites in small-volume biological samples. The assay was linear over the therapeutically relevant concentration range (0.125–10,000 nM); in brain tissue, the lower limit of quantification was 0.247 nM and the sensitivity was 1.4 pg. The ability to quantify DXR and detect metabolite formation may provide insight into the toxicity and bioavailability of drug incorporated into carriers such as liposomes.

Liquid‐phase microextraction combined with liquid chromatography‐mass spectrometry. Extraction from small volumes of biological samples

AbstractLiquid‐phase microextraction (LPME) is a sample preparation technique based on disposable polypropylene hollow fibres, which results in efficient sample clean‐up and high preconcentration. The present paper describes the combination of LPME with LC‐MS utilising electrospray ionisation for high sensitivity. Nine antidepressant drugs were extracted from 50 or 500 μL of plasma or whole blood samples, through a thin layer of dodecyl acetate immobilised in the pores of the hollow fibre, and into 15 μL of 200 mM formic acid as acceptor solution inside the hollow fibre. Analyte recoveries in the range 12–68% and 9–52% were obtained from 50 μL of plasma and whole blood respectively. The acceptor solution (15 μL) was diluted with 60 μL of 5 mM ammonium formate pH = 2.7 prior to injection into the LC‐MS system. The system was qualitatively investigated for matrix effects utilising a post‐column infusion system. Whole blood from 5 different persons was cleaned‐up by LPME and injected onto the analytical column while a solution of the 9 model compounds was continuously infused post‐column. No signs of ion suppression were seen for any of the model compounds. Limits of quantification (S/N = 10) were in the low ng/mL range for 6 of the 9 model compounds utilising a whole blood sample volume of only 50 μL. The repeatability of the extractions was investigated utilising paroxetine as internal standard. Acceptable RSDs (%) were obtained (&lt; 20%) for 5 of the antidepressants. By increasing the sample volume from 50 to 500 μL of whole blood RSDs below 20% (3–16%) were observed for all 8 antidepressants.

Comprehensive phenotyping and biological marker discovery.

There is an enormous unmet need for biological markers to characterize disease type, status, progression, and response to therapy. We are developing and applying an integrated bioanalytical platform and clinical research program to facilitate comprehensive differential phenotyping of patient samples and enable the discovery of biomarkers. The platform employs high-throughput, quantitative analysis for the characterization of thousands of parameters including cell populations, cell-surface antigen density, soluble proteins and soluble low molecular weight biomolecules, from small-volume biological samples in a clinical research laboratory-like setting.

Analysis of human urine protein biomarkers via biomolecular interaction analysis mass spectrometry

Biomolecular interaction analysis mass spectrometry (BIA/MS) is a two-dimensional chip-based analytical technique geared toward quantitative and qualitative analysis of small volumes of biological samples. Interactions between surface-immobilized ligands and solute-borne analytes are quantitatively viewed in real time through surface plasmon resonance sensing, followed by qualitative matrix-assisted laser desorption/ionization time-of-flight MS analysis of the analyte(s) affinity-retained on the sensor surface. In this work, BIA/MS was used in the detection of a number of protein biomarkers from human urine. Small volumes of human urine were analyzed for cystatin C, β2-microglobulin, urinary protein 1, and retinol-binding protein (RBP). Multiaffinity sensor surfaces were created to simultaneously and rapidly detect all four proteins in a single BIA/MS analysis on a two-flow cell sensor chip configuration. Furthermore, RBP was analyzed separately from both urine and plasma samples. Results indicate that BIA/MS can be used successfully in rapid screening of a number of urinary proteins indicated as putative biological markers for renal dysfunction. © 2001 by the National Kidney Foundation, Inc.

Quantification of metronidazole in small-volume biological samples using narrow-bore high-performance liquid chromatography

A rapid, selective and sensitive HPLC assay has been developed for the routine analysis of metronidazole in small volumes of rat plasma, gastric aspirate and gastric tissue. The extraction procedure involves liquid–liquid extraction and a protein precipitation step. A microbore Hypersil ODS 3 μm (150×2.1 mm I.D.) column was used with a mobile phase consisting of acetonitrile–aqueous 0.05 M potassium phosphate buffer (pH 7) containing 0.1% triethylamine (10:90). The column temperature was at 25°C and the detection was by UV absorbance at 317 nm. The limit of detection was 0.015 μg ml −1 for gastric juice aspirate and plasma and 0.010 μg g −1 for gastric tissue (equivalent to 0.75 ng on-column). The method was linear up to a concentration of 200 μg ml −1 for plasma and gastric juice aspirate and up to 40 μg g −1 for tissue, with inter- and intra-day relative standard deviations less than 14%. The measured recovery was at least 78% in all sample matrices. The method proved robust and reliable when applied to the measurement of metronidazole in rat plasma, gastric juice aspirate and gastric tissue for pharmacokinetic studies in individual rats.

Microdevices for Biological Analyses Recent Advances and Directions for the Future

SAMPLE ACQUISITION AND CONTROL Biological sampling is important for drug discovery applications and for the basic biochemical and neurochemical discovery process. Several issues arise when sampling from biological environments. First, important biology can occur in small volumes (nL-pL) and at low concentrations (mM-nM). In order to access this information, the important biochemicals must be detectable and the time of specific biochemical events must be recorded. This means that a sampling and analysis method should be selective for the analyte(s) of interest, without loss or dilution, for efficient analysis. Tremendous accomplishments in biological sampling and analysis have been made, both for sampling and microfluidic manipulation. This article focuses on advances in controlling small volumes of biological samples and the analysis of specific analytes in those samples.

Microdevices for Biological Analyses Recent Advances and Directions for the Future

SAMPLE ACQUISITION AND CONTROL Biological sampling is important for drug discovery applications and for the basic biochemical and neurochemical discovery process. Several issues arise when sampling from biological environments. First, important biology can occur in small volumes (nL-pL) and at low concentrations (mM-nM). In order to access this information, the important biochemicals must be detectable and the time of specific biochemical events must be recorded. This means that a sampling and analysis method should be selective for the analyte(s) of interest, without loss or dilution, for efficient analysis. Tremendous accomplishments in biological sampling and analysis have been made, both for sampling and microfluidic manipulation. This article focuses on advances in controlling small volumes of biological samples and the analysis of specific analytes in those samples.

Determination of carbohydrates as their dansylhydrazine derivatives by capillary electrophoresis with laser-induced fluorescence detection.

A mixture of mono- and disaccharides was derivatized with dansylhydrazine in a relatively fast chemical reaction (15 min) which is selective toward aldehydes and ketones. The products of the derivatization reaction were then separated by capillary electrophoresis (CE) using laser-induced fluorescence (LIF) as the detection mode. The smallest amount of carbohydrate derivatized and determined by CE/LIF was 10 pmol contained in 100 microL of the final reaction mixture. The CE/LIF method provided a linear response for the carbohydrates tested (over 2-3 orders of magnitude), with a limit of detection of 100 amol. Separation efficiencies over 250,000 theoretical plates were obtained. The procedure was used to analyze a tear fluid sample to demonstrate the applicability of the method for the glucose determination in small volumes of biological samples.

Characterization of Absorption and Scattering Properties of Small-Volume Biological Samples Using Time-Resolved Spectroscopy

With time-resolved spectroscopy, we develop an experimental approach by sample substitution to measure the absorption (μa) and reduced scattering (μ′s) coefficients of small-volume biological samples. To investigate the method, small-volume control samples are substituted into a large-size host medium during increases in the absorber (or scatterer) concentration of the host. By characterizing the deviation of the spectra taken with and without the sample, we determine the matching points where the sample and surrounding medium are optically identical. We show that this method can result in correct values of the μa and μ′s for the sample within 6% error if the matching conditions for both the μa and the μ′s are fully realized. The results also indicate that this method can give approximate values of the μa and μ′s in a reasonable range if either the μa or the μ′s matching between the two media is realized. This method has been applied to the studies of absorption properties of a human finger and of scattering properties of yeast.

Electrochemical cells for voltammetry, coulometry, and protein activity assays of small-volume biological samples

Cell designs, experimental protocols, and results for electrochemical investigation of small quantities of biological materials under anaerobic conditions are reported. Three types of electrochemical experiments are considered: (i) cyclic voltammetry of 20- to 100-μl samples; (ii) direct coulometry of 0.5- to 1.5-ml samples; and (iii) an electrochemically initiated protein activity assay which includes provision for analysis of gaseous reaction products and correlation with electron flux. The first two procedures are illustrated by measurement of the formal electrode potential ( E o′) and number of electrons transferred ( n) in redox reactions of small quantities of biological and inorganic materials. The third procedure is illustrated by assaying the activity of the MoFe protein plus Fe protein complex from Azotobacter vinelandii nitrogenase for reduction of C 2H 2 to C 2H 4.