High Analyte Concentrations Research Articles

Isotope ratio precision with transient sample introduction using ICP orthogonal acceleration time-of-flight mass spectrometry

An assessment of inductively coupled plasma orthogonal acceleration time-of-flight mass spectrometry (ICP-oa-TOF-MS) for measurement of isotope ratios arising from steady-state and transient signal detection is presented. An HPLC pump and autosampler were used to introduce microliter volumes of analyte solution into a carrier flow to generate precise and repeatable transient signals having a FWHM of approximately 1 s. Analog and pseudo pulse data processing modes were evaluated and found to obey Poisson statistics for prediction of isotope ratio performance. Use of pseudo pulse counting is restricted by the limited linear range of response and cannot exceed approximately 0.2 counts per pushout. This limitation does not impede analog detection, which thus permits precise isotope ratio measurements to be achieved at higher analyte concentrations using shorter integration times. For transient signals, area measurements are superior to averaging point-to-point measurements across the peak for optimum ratio precision. Although the TOF system provides for simultaneous sampling of the extracted pulse, thereby permitting elimination of correlated noise through ratioing techniques, detection remains sequential and noise components present in the detection system limit isotope ratio precision in this study to 0.04% RSD.

Liquid chromatography coupled to nuclear magnetic resonance spectroscopy for the identification of isoflavone glucoside malonates in T. pratense L. leaves.

Previous studies revealed that the main isoflavones in extracts of leaves of T. pratense L. are biochanin A and formononetin, their 7-O-glucosides, and two glucoside malonate isomers of each of them. Since LC-MS(/MS) did not provide sufficient information to distinguish the glucoside malonate isomers, in the present paper LC-NMR as well as off-line two-dimensional NMR were used to obtain further structural information. Matrix solid-phase dispersion (MSPD) was applied to obtain sufficiently high analyte concentrations to perform LC-NMR. Stop-flow reversed-phase LC-NMR was performed using a gradient of deuterated water and deuterated acetonitrile. Offline COSY and NOESY experiments were carried out to determine the positions of the glucose moiety on the flavonoid aglycone, and of the malonate moiety on the glucose. Based on the fragmentation patterns in MS/MS and the NMR spectra, the two formononetin glucoside malonate isomers were identified as 7-O-beta-D-glucoside 6"-O-malonate and 7-O-beta-D-glucoside 4"-O-malonate; i.e. they only differ in the substitution position of the malonate group on the glucoside ring. The biochanin A glucoside malonate isomers, however, have quite different structures. The main and later eluting isomer is biochanin A 7-O-beta-D-glucoside 6"-O-malonate, and the minor and earlier eluting isomer is 5-hydroxy-7-methoxyisoflavone 4'-O-beta-D-glucoside 4"-O-malonate: the positions of the methoxy group and the glucoside 6"-O-malonate group on the flavonoid skeleton are interchanged.

Matrix-dependent cationization in MALDI mass spectrometry.

The matrix dependence in cationization processes, the competition between cationization and protonation and the question of whether gas-phase cation transfer or attachment of free cations dominates in matrix-assisted laser desorption/ionization mass spectrometry were studied. Two different sample preparation methods were employed, the dried-droplet sample preparation and a mixture of solid matrix, analyte and salt. The latter ensures that the formation of cation adducts takes place in the gas phase. By monitoring the suppression of matrix signals for different matrices, it was found that matrices with high gas-phase metal ion binding energies require high analyte concentrations for matrix suppression to occur. By comparing the mass spectra obtained using sinapinic acid or sinapinic methyl ester as a matrix, a correlation between cationization and deprotonation of matrix molecules was found. It is also demonstrated that attachment of free gas-phase cations, rather than cation transfer from the cationized matrix, is the predominant process in cationization.

High-sensitivity methods for sequential injection determination of the main constituents of alloys

A sequential injection system with spectrophotometric detection was proposed for the determination of copper, zinc and lead in brass. In view of the high analyte concentrations and the sensitivity of the involved catalytic methods, optimization of the flow system was carried out aiming mild reaction conditions. Copper and lead determinations were based on the catalytic effects of Cu 2+ and Pb 2+ on the oxidation rates of resorcinol and pyrogallol red by H 2O 2, respectively, and zinc determination involved precipitate formation after oxidation of 1-nafthylethylenediamine by K 3Fe(CN) 6. The analytical procedures were designed with some common parameters such as pH=9.0 (borate system), λ=530 nm, and H 2O 2 as oxidizing agent; system geometry was maintained. The proposed system is rugged, and base line drift is not observed during 4 h operation periods. Twenty samples are run per hour (60 determinations) and reagent consumption is minimal, thus avoiding drawbacks related to waste management. Precise results (R.S.D.<1.0%; n=7) are obtained and a detection limit of 1% (w/w) was estimated for the three analytes. Results were in agreement with flame atomic absorption spectrometry.

Attoliter detection volumes by confocal total-internal-reflection fluorescence microscopy.

We report a confocal total-internal-reflection fluorescence (TIRF) microscope that generates a detection volume for analyte molecules of less than 5 al (5 x 10(-18) l) at a water-glass interface. Compared with conventional confocal microscopy, this represents a reduction of almost 2 orders of magnitude, which is important in isolating individual molecules at high analyte concentrations, where many biologically relevant processes occur. Diffraction-limited supercritical focusing and fluorescence collection is accomplished by a parabolic mirror objective. The system delivers TIRF images with excellent spatial resolution and detects single molecules with a high signal-to-background ratio.

HPLC determination of erythrocyte methotrexate polyglutamates after low-dose methotrexate therapy in patients with rheumatoid arthritis.

Methotrexate (MTX) may produce antiarthritic effects through polyglutamation to methotrexate polyglutamates (MTXPGs), a process that covalently attaches sequential gamma-linked glutamic residues to MTX. We sought to develop an innovative HPLC method for the quantification of these metabolites in erythrocytes. Two alternative approaches were developed. In the first approach, MTXPGs from 50 micro L of packed erythrocytes were converted to MTX in the presence of plasma gamma-glutamyl hydrolase and mercaptoethanol at 37 degrees C. In the second approach, MTXPG species (up to the hepta order of glutamation) from 100 micro L packed erythrocytes were directly quantified in a single run. In both methods, the MTXPGs were extracted from the biological matrix by a simple perchloric acid deproteinization step with direct injection of the extract into the HPLC. The chromatography used a C(18) reversed-phase column, an ammonium acetate/acetonitrile buffer, and postcolumn photo-oxidation of MTXPGs to fluorescent analytes. Intra- and interday imprecision (CVs) were <10% at low and high concentrations of analytes for both methods. The limit of quantification was 5 nmol/L. In 70 patients with rheumatoid arthritis receiving weekly low-dose MTX, the mean (SD) total MTXPG concentration measured after conversion of MTXPGs to MTX was similar to the total MTXPG concentration calculated from the sum of individual MTXPG species [117 (56) vs 120 (59) nmol/L; r = 0.97; slope = 1.0]. The triglutamate predominated over all other MTXPG species (36% of total), the pentaglutamate was the highest order of glutamation detected, and a stability study revealed no change in the polyglutamation pattern in erythrocytes 48 h after phlebotomy when the specimen was stored at 2-8 degrees C. The proposed method for quantification of erythrocyte MTXPGs is rapid, sensitive, and accurate and can be applied to the routine monitoring of MTX therapy.

A mathematical model of lateral flow bioreactions applied to sandwich assays

Lateral flow (LF) biodetectors facilitate low-cost, rapid identification of various analytes at the point of care. The LF cell consists of a porous membrane containing immobilized ligands at various locations. Through the action of capillary forces, samples and reporter particles are transported to the ligand sites. The LF membrane is then scanned or probed, and the concentration of reporter particles is measured. A mathematical model for sandwich assays is constructed and used to study the performance of the LF device under various operating conditions. The model provides insights into certain experimental observations including the reduction in the level of the detected signal at high target analyte concentrations. Furthermore, the model can be used to test rapidly and inexpensively various operating conditions, assist in the device’s design, and optimize the performance of the LF device.

Effect of Weak Electrolytes on Electromigration Dispersion in Capillary Zone Electrophoresis

In this paper, simple equations are introduced to predict the electromigration dispersion (EMD) in weak background electrolytes (BGEs) at high pH in capillary zone electrophoresis (CZE) separations. Analytical solutions have been developed in the case of a basic BGE constituted of a neutral acid in equilibrium with its anionic conjugate base, which acts as co-ion to the analytes, and the treatment has been extended to other buffering systems. The treatment assumes that the concentration of the neutral form of the buffer species remains constant throughout the separation and that the pair of ionic species, hydroxide ion and co-ion, effectively act as a single co-ion. Hydroxide ion is shown to have a significant effect on EMD for pH values ≥10. The change in hydroxide ion concentration in the sample zone can compensate for changes in conductivity due to the difference of mobility between the analyte and the co-ion. Under appropriate conditions, the hydroxide species can correct the distortion due to the difference of mobility, and a good peak shape can be obtained, even with a high analyte concentration. This effect has been modeled using numerical treatments and experimentally validated using benzoate and 4-hydroxybenzoate as test analytes in a BGE constituted of a 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) buffer with sodium as counterion. At constant co-ion concentration, the peak efficiency improved from 5.1 × 104 to 1.3 × 105 by changing the pH from 10.85 to 11.00. At a constant pH of 10.94, the peak distortion reversed in sign on increasing the concentration of CAPS co-ion from 15 to 25 mM. All experimental electropherograms were successfully fitted using the Haarhoff−Van der Linde function and distortion parameters extracted. Experimental and predicted distortion parameters are in good agreement. This work offers the potential to improve peak shapes in CZE by taking into account the effect of an ion involved in the water autoprotolysis equilibrium, and could lead to improvement in formulation of low and high pH buffers for CZE.

Time-weighted average passive sampling with a solid-phase microextraction device.

A modified Solid-Phase Microextraction (SPME) device has been used as a passive sampler to determine the time-weighted average (TWA) concentration of volatile organic compounds (VOCs) in air. Unlike conventional sampling with SPME, in which the fiber is extended outside its needle housing, during TWA passive sampling, the fiber is retracted a known distance into its needle housing. The SPME passive sampler collects the VOCs by the mechanism of molecular diffusion and sorption on to a coated fiber as collection medium. This process has been shown to be described by Fick's first law of diffusion, whereby determination of the amounts of analytes accumulated over time enable measurement of the TWA concentration to which the sampler was exposed. A series of fibers, 100-microm poly(dimethylsiloxane), 65-microm poly(dimethylsiloxane)/divinylbenzene, and 75-microm Carboxen/poly(dimethylsiloxane), were tested for their "zero sink", face velocity, and response time behavior. Of the fibers tested, that coated with 75-microm Carboxen/poly(dimethylsiloxane) was found to be an excellent passive sampler for VOCs. TWA passive sampling with a SPME device was shown to be almost independent of face velocity and to be more tolerant of high and low analyte concentrations and long and short sampling times, because of the ease with which the diffusion path length could be changed. It was found that environmental conditions, e.g., temperature, pressure, relative humidity, and ozone, have little or no effect on sampling. The 75-microm Carboxen/poly(dimethylsiloxane) fiber can retain VOCs for up to two weeks without significant loss. When the SPME device was tested in the field and the results were compared with those from National Institute of Occupational Health and Safety method 1501, good agreement was obtained.

Characteristics of atomic absorption calibration curves with the transversely heated graphite furnace

Calibration curves with two quasi-linear sections (“double sloping”) were observed for the medium volatile elements, Cr and Cu, with the use of a SIMAA 6000 graphite furnace atomic absorption spectrometer under interrupted internal gas flow conditions. If a standard transversally heated graphite atomizer (THGA) tube was shortened by 0.5 mm at both of its ends, (i.e. the gaps were enlarged between graphite furnace housing and tube ends), a stronger declination of the calibration curves resulted. Elements with fairly high diffusion coefficients (>5.8 cm2 s−1) and with short appearance time of their transients (<0.6 s), such as Cr and Cu, have shown the most characteristic sensitivity drop towards higher concentrations. This anomalous feature could be eliminated in three different ways; (1) by applying end-capped THGA tubes, (2) using mini-flow (50 cm3 min−1) conditions during the atomization stage, and (3) by adding Pd–Mg chemical modifier. For the low volatile Mo and V, the calibration curves had no irregular shape. For Ag, Co, Cr, Cu, Mn and Ni, the mini-flow settings improved the linearity of the calibration curves and extended the upper limit of the linear calibration range by a factor of 1.5–2.0. The irregular characteristic of the analytical curves was interpreted as an increased vapour loss at higher analyte concentrations through the opened ends of the standard THGA tubes. This vapour loss was associated with the significantly diverse expulsion velocities of atoms, caused by the difference in temperature and concentration gradients, when evaporating amounts of analytes with more than one order of magnitude difference.

Statistical Evaluation of Analytical Methods for the Determination of Rare-Earth Elements in Geological Materials and Implications for Detection Limits

We briefly describe the analytical methods commonly employed for determining rare-earth elements (REE) in geological materials. A compilation of analytical data for 24 International Geochemical Reference Materials (IGRM) from the United States and Japan is used to evaluate statistically the inter-laboratory performance of various methods. The most frequently employed method groups for measuring REE include mass spectrometry (MS), nuclear methods (NM), and emission spectrometry (ES), although separation methods (SM) such as high-performance liquid chromatography (HPLC), ion chromatography (IC), or capillary electrophoresis (CE) have shown great potential as cheap, rapid, precise, and accurate methods. X-ray fluorescence (XRF), atomic absorption (AA), and classical colorimetric (CL) methods are not generally recommended for the determination of REE in geological materials, unless suitable pre-concentration procedures are used. The initial inter-laboratory REE data are generally positively skewed due to the presence of mostly high analyte concentration outliers. After an appropriate statistical rejection of outlying observations, the remaining data sets for individual groups of methods show that MS provides the most precise REE data. Inter-laboratory detection limits obtained by weighted regression of a linear precision model are generally in the sub-ppm range (0.07-1.5 ppm for MS methods; 0.05-3.1 ppm for NM; 0.1-4.2 ppm for ES). No overall significant bias was found among the MS, NM, and ES groups of methods for the analysis of the REE, but the Student's t-test revealed significant differences (even at a strict significance level of 1%, equivalent to a confidence level of 99%) for some REE in a few IGRM (for MS-NM methods, La in granite JG-1, Ce in diabase W-1, Tb in basalt BHVO-1, Dy in andesite AGV-1, Tm in basalt BCR-1, and Lu in basalt BIR-1; for MS-ES methods, La in rhyolite JR-1). For these cases, the analytical data must be treated separately to define mean concentration values for methods and to assign one of the method means (probably from the most precise method MS, NM, or ES) as the mean value of that element in the IGRM. The average concentration data from well-established methods can be successfully used to evaluate the performance of other analytical methods such as SM and XRF, not in general use for determination of REE. Finally, the method detection limits obtained for inter-laboratory data, as well as for individual laboratories are shown, for the first time, to depict a zigzag pattern, obeying the well-known "odd-even" effect of nuclear stability that governs element concentrations in the solar system and the abundance of individual isotopes. We show that the REE detection limits for all analytical methods mimic completely the zigzag patterns for actual REE concentration data in all kinds of geological and cosmological materials, and we hypothesize, on this basis, that the analytical detection and quantification process is also governed by the same nuclear "odd-even" effect.

A straightforward means of coupling preparative high-performance liquid chromatography and mass spectrometry

Flow splitting to a mass spectrometer is a common way of coupling a highly specific detector to preparative or semi-preparative high-performance liquid chromatography (HPLC) purification of combinatorial libraries, drug metabolites, and characterizable impurities. The sensitive mass spectrometer consumes only a small fraction of the analyte while providing online structure-specific detection, and its output can thus be used to trigger collection of the desired fraction. Coupling mass spectrometry to preparative HPLC is difficult due to the susceptibility of the detector to fouling under conditions of high analyte concentration or solute amount, or to changes in solvent composition. We report here on a device, the mass rate attenuator (MRA), which automatically produces split ratios over a range of 100:1 to 100 000:1 under programmable user control. The MRA is a flow-control device that periodically gates a small aliquot from one liquid stream into another. The design allows the user to set the frequency of the gating without interruption of the HPLC flow stream. The MRA also allows control of the volume of the aliquot that is transferred between the flow streams. This additional control, compared to passive splitting devices, facilitates optimization of the tubing connecting the separation, detection and collection events. We demonstrate that such optimization can reduce the volume of the collected fraction without compromising recovery, thus reducing the time spent in evaporating solvents to reclaim purified products. Copyright © 2002 John Wiley & Sons, Ltd.

A straightforward means of coupling preparative high-performance liquid chromatography and mass spectrometry.

Flow splitting to a mass spectrometer is a common way of coupling a highly specific detector to preparative or semi-preparative high-performance liquid chromatography (HPLC) purification of combinatorial libraries, drug metabolites, and characterizable impurities. The sensitive mass spectrometer consumes only a small fraction of the analyte while providing online structure-specific detection, and its output can thus be used to trigger collection of the desired fraction. Coupling mass spectrometry to preparative HPLC is difficult due to the susceptibility of the detector to fouling under conditions of high analyte concentration or solute amount, or to changes in solvent composition. We report here on a device, the mass rate attenuator (MRA), which automatically produces split ratios over a range of 100:1 to 100 000:1 under programmable user control. The MRA is a flow-control device that periodically gates a small aliquot from one liquid stream into another. The design allows the user to set the frequency of the gating without interruption of the HPLC flow stream. The MRA also allows control of the volume of the aliquot that is transferred between the flow streams. This additional control, compared to passive splitting devices, facilitates optimization of the tubing connecting the separation, detection and collection events. We demonstrate that such optimization can reduce the volume of the collected fraction without compromising recovery, thus reducing the time spent in evaporating solvents to reclaim purified products.

Reporter Genes lucFF, luxCDABE, gfp, and dsred Have Different Characteristics in Whole-Cell Bacterial Sensors

The selection of a genetic reporter can be difficult because of the wide range of genes available. In order to reduce the selection, we compared the performance of different reporter genes: firefly luciferase (Photinus pyralis lucFF), bacterial luciferase operon (Photorhabdus luminescens luxCDABE), green fluorescent protein (Aequorea victoria gfp), and red fluorescent protein (Discosoma sp. dsred) in whole-cell bacterial sensors. Escherichia coli sensor bacteria were engineered to contain a reporter plasmid that carries the reporter gene under the control of mercury- (mer from Tn21) or arsenite- (ars from R773) responsive regulatory units. Characteristics of the strains were studied by using different arsenite or mercury concentrations and incubation times. The lowest detectable concentration of analytes and the fastest responses were achieved with lucFF or luxCDABE as reporter genes. The fluorescent proteins, GFP and DsRed, gave responses at higher analyte concentrations and after significantly longer incubation times. The results indicate that luciferases are better reporters in whole-cell sensor bacteria.

Extending the solid-phase microextraction technique to high analyte concentrations: measurements and thermodynamic analysis.

The solid-phase microextraction (SPME) technique has been used historically to quantify analytes present at the parts per million level. However, the nonintrusive nature of SPME lends itself to other applications involving analytes at higher concentration. In the current work, the possibility of using the SPME technique to measure concentrated gaseous samples was examined. Pentane concentrations between 0 and 100% saturation were studied, over a temperature range of 20-45 degrees C. The results showed that, up to a critical mole fraction in the solid phase, the concentrations of pentane in the polymeric extracting solid and vapor phases were related by a constant, equal to Henry's constant. The temperature dependence of Henry's constant was shown to follow the predicted trend with temperature, as determined from rigorous thermodynamic calculations. Above the pentane concentration in the polymeric phase, the response deviated from linearity. The nonideality was captured in an activity coefficient. An activity coefficient model developed to describe the nonideality was found to be a function of the swollen volume of the SPME polymer phase. The results indicate that the SPME technique can be applied to high analyte concentrations, although difficulties may be encountered when multiple analytes are absorbed.

HPLC Determination of Thiopurine Nucleosides and Nucleotides in Vivo in Lymphoblasts following Mercaptopurine Therapy

Mercaptopurine is a prodrug requiring intracellular activation to thiopurine nucleotides to exert antileukemic effect. We developed a reversed-phase liquid chromatographic assay for the quantification of mercaptopurine, thioguanine, and methylmercaptopurine nucleoside and nucleotide concentrations in the target tissue, the leukemic lymphoblast. Leukemic blasts were isolated from peripheral blood and bone marrow by a standard Ficoll-hypaque procedure. Proteins were removed by ultrafiltration in the presence of dithiothreitol. Thiopurine ribonucleotides were converted into their respective ribonucleosides by treatment of ultrafiltrate with acid phosphatase. Thiopurine nucleosides and bases were measured by direct injection of ultrafiltrate into the chromatographic system. Thiopurine nucleotide concentrations were calculated by subtracting the thiopurine nucleoside concentrations measured after treatment with acid phosphatase from those measured after direct injection of ultrafiltrate in the chromatographic system. Analytes were separated on a C18 Supelco column with ammonium phosphate-methanol eluent coupled with ultraviolet detection. CVs for intra- and interday precision were 1.1-14% (median, 4.9%), and recovery of added analyte was 89-126% (median, 105%) at low and high concentrations of analytes, except for mercaptopurine riboside. The median signal for each of the five metabolites in lymphoblast samples was 98% (range, 80-106%) of that in water. Detection limits for thiopurine bases and nucleosides ranged from 0.5 to 4.5 pmol/5 x 10(6) cells. This method is suitable for measurement of thiopurine metabolite concentrations in lymphoblasts in children with acute lymphoblastic leukemia following a single dose of intravenous mercaptopurine.

HIGH DOSE HOOK EFFECT IN SERUM TOTAL AND FREE PROSTATE SPECIFIC ANTIGEN IN A PATIENT WITH METASTATIC PROSTATE CANCER

High dose hook effect is a phenomenon in which samples with extremely high analyte concentrations may paradoxically produce a lower response than expected in an immunoradiometric assay. This effect has been observed while measuring serum prostate specific antigen (PSA) with a Tandem R assay (Hybritech Beckman-Coulter Corp., San Diego, California). 1 We report the occurrence of a hook effect while measuring total and free PSA in a patient with metastatic prostate cancer. CASE REPORT An 85-year-old man was referred to us for paraplegia and suspected bone metastasis of prostate cancer. Serum total and free PSA measured with a Tandem R assay before digital rectal examination was 11 and 0.5 ng./ml., respectively, and g-seminoprotein measured with an Eiken kit (Eiken, Tokyo, Japan) was 1,800 ng./ml. Digital rectal examination revealed enlargement of the left lobe with no extension beyond the prostate. Bone scintigraphy suggested metastases to the pelvis with the extent of disease score 2. Pathological examination confirmed the diagnosis of adenocarcinoma of the prostate (Gleason 4 to 5). Orchiectomy was performed simultaneously. Because we thought total PSA of 11 ng./ml. was falsely low, repeat analysis after dilution was performed. After 1,0003 dilution total and free PSA concentrations were 26,000 and 1,050 ng./ml., respectively. We determined that the hook effect had occurred in this case. Although total and free PSA values decreased, the patient died of prostate cancer 2 months after orchiectomy. DISCUSSION

Adaptation of a surface plasmon resonance biosensor with microfluidics for use with small sample volumes and long contact times.

The efficient delivery of sample to surface-immobilized sites is a key element in biosensing. For a surface plasmon resonance (SPR) biosensor, this has been addressed by constant flow through a microfluidic system with a sample injection loop (Sjölander, S.; Urbaniczky, C. Anal. Chem. 1991, 63, 2338-2345). The present study describes an alternative mode of sample delivery without constant unidirectional flow. It was implemented on a commercial Biacore X SPR biosensor equipped with a microfluidic cartridge, but with the fluidic handling performed by an externally computer-controlled syringe pump. We demonstrate that sample volumes as low as 2 microL can be reproducibly positioned to cover the sensor surfaces, manipulated in a serial fashion, efficiently mixed by applying an oscillatory flow pattern, and fully recovered. Compared to the traditional continuous unidirectional flow configuration, we found very similar kinetic responses at high analyte concentrations and slightly slower responses at low concentrations, most likely due to depletion of analyte from the small sample volumes due to surface binding. With the antibody-antigen systems tested, binding parameters were obtained that are generally within 10% of those from conventional experiments. In the new configuration, biosensor experiments can be conducted without the usual constraints in the surface contact time that are correlated with sample volume and mass transport rate. This can translate to improved detection limits for slow reactions and can facilitate kinetic and thermodynamic binding studies.

Nanoliter solvent extraction combined with microspot MALDI TOF mass spectrometry for the analysis of hydrophobic biomolecules.

A nanoliter solvent extraction technique combined with microspot matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is presented. This method involves the use of a nanoliter droplet containing organic solvents at the tip of a small capillary for extraction. The droplet is formed inside a microliter aqueous sample containing the analyte of interest. After extraction, the droplet is deposited onto a MALDI target precoated with a thin matrix layer. Since the nanoliter droplet never touches the sample container wall, any possible extraction of contaminants adsorbed on the plastic or glassware is avoided. In addition, there is no need to concentrate the organic phase after the extraction, thus avoiding any possible loss during the concentration step. The nanoliter volume can be readily deposited onto a MALDI target, producing a high analyte concentration within a microspot. Combined with microspot MALDI, this technique allows for very sensitive analysis of the extracted analyte. The performance of this technique is illustrated in several applications involving the detection of hydrophobic peptides or phospholipids. It is shown that very hydrophobic analytes can be extracted from small-volume samples containing a large amount of salts and/or more hydrophilic analytes, which tend to give dominant signals in conventional MALDI experiments. Nanoliter extraction of analyte from samples containing less than 100 nM hydrophobic analyte and over 1 microM easily ionized hydrophilic species is demonstrated. Finally, using the analysis of the ionophore valinomycin as an example, it is demonstrated that the technique is a more reliable tool for probing metal-peptide complexes than regular MALDI sample preparations.

A Duplexed Microsphere-Based Fluorescent Immunoassay

Microsphere-based immunoassays are described for the simultaneous measurement of the clinically important drugs digoxin and theophylline. Competitive immunoassays were performed using haptenized microspheres and antibodies labeled with horseradish peroxidase. Enzyme-catalyzed reporter deposition (CARD) resulted in immunofluorescence signal amplification. Two encoding dyes were used to differentiate analytical signals from microspheres containing assays for the two analytes. An epifluorescence microscope and a CCD camera interfaced with a computer were utilized to measure fluorescence signals of individual microspheres. The microspheres from a duplexed assay were mounted on microscope slides as well as inserted into wells etched into the distal ends of optical imaging fibers. Fluorescence images from both formats were captured. In the experiments using microscope slides, the immunoassays were successfully duplexed and only marginal interferences at high analyte concentrations were observed. Preliminary results suggest that simultaneous determination of the two analytes using a fiber-based sensor-array format is feasible, but requires further development before precise quantitative analyses are possible.