Chip-based capillary electrophoresis/mass spectrometry determination of carnitines in human urine

A chip-based capillary electrophoresis/mass spectrometry (CE/MS) system is described for the CE separation and on-line electrospray detection of carnitine and selected acylcarnitines from mixtures of analytical standards as well as extracts of fortified human urine. Chip-based CE/MS experiments in two different laboratories were carried out using a triple-quadrupole mass spectrometer and a quadrupole time-of-flight (QTOF) mass spectrometer, respectively. The glass chips used with both systems were comparably equipped with a microfabricated capillary electrophoresis (CE) channel but with different electrosprayers. The quadrupole chip-based CE/MS experiments employed a miniature coupled microsprayer, which allowed coupling of the microelectrospray process via a micro liquid junction at the exit of the CE capillary channel. Selected ion monitoring (SIM) CE/MS experiments were employed for all of the quadrupole CE/MS work. The QTOF CE/MS full-scan single MS and MS/MS experiments were carried out in another laboratory using accurate mass measurement TOF mass spectrometry techniques. The electrospray process that was employed with the QTOF system differed in that an inserted nanoelectrospray capillary needle was carefully affixed into a flat-bottomed hole that was aligned with the CE channel exit orifice. SIM CE/MS using the described quadrupole system provided acceptable ion current electropherograms from fmole levels from analytical standard solutions of carnitine and acylcarnitines that were manually injected (loaded) onto the chip. In addition, the corresponding electropherograms for human urine fortified with the target carnitine and acylcarnitines at a 10-20 microg/mL (35-124 microM) level were obtained via SIM CE/MS techniques. The measured CE separation efficiency for the SIM CE/MS electropherograms was determined to be 2860 plates (peak width at half-height method or N = 5.54(T/WO.5(2)), and carnitine and three acylcarnitines were separated in less than 48 s. In contrast, using quadrupole-TOF technologies, the same samples could be diluted by a factor of 2-4 to obtain a comparable detector response for the target compounds. In the full-scan, single mass analyzer mode (m/z 150-500), the CE separation efficiency was measured to be 2600 plates, but mass measurement accuracy was less than 5.0 ppm for the quaternary cations. In the CE/MS/MS mode, full-scan collision-induced dissociation (CID) mass spectra were obtained with a mass accuracy of < or =10 ppm for the higher mass ions and < or =27 ppm for the lower mass product ions. These results demonstrate the feasibility for on-chip CE separation and electrospray mass spectrometric detection for these important compounds in synthetic mixtures, as well as in human urine extracts.     

Electrospray device for coupling microscale separations and other miniaturized devices with electrospray mass spectrometry

A miniaturized ion sprayer device is described which is suitable for coupling with chip-based analytical separation devices, multiwell plates, or surfaces containing residues of prepared samples. Two versions of a similar device are described. A "microsprayer" device suitable for coupling to the terminal edge of a capillary electrophoresis (CE) chip is constructed from modified 1/16-in. HPLC fittings. This microsprayer employs a free-standing liquid junction formed via continuous delivery of a flow (2-6 microL/min) of suitable solvent which carries the CE effluent through a pneumatically assisted electrospray (ion spray) needle positioned in front of an atmospheric pressure ionization (API) mass spectrometer. A related but larger "minisprayer" device is also described which employs the same features as the microsprayer, but with an extended sampling capillary tube which can reach into the depths of 96-, 384-, and 1536-multiwell plates containing either sample solutions or dried sample residues. The minisprayer may be positioned in front of an API ion sampling orifice and the multiwell plate positioned stepwise from sample to sample for analysis of trace samples contained in the wells. The resulting infusion-ion spray mass spectrometric analyses can provide sequential analysis of previously prepared biological samples containing small drug compounds, proteins, and related compounds. This same device is also shown to be useful for sampling from a surface containing trace level compounds of biological interest. Results are shown that demonstrate microscale separations and selected ion monitoring (SIM) capillary electrophoresis/mass spectrometry (CE/MS) detection of berberine and palmatine using the microsprayer. SIM ion spray determination of a 2 ng/microL solution of berberine contained as a dry residue in the bottom of a 384-well plate as well as full-scan electrospray mass spectra for low-picomole levels of cytochrome c contained in a 1536-well microtiter plate are shown. The respective micro- and minisprayer devices provide a simple yet effective means of transferring trace-level samples either from a microscale or chip-based separation device as well as samples contained in multiwell plates which are increasingly employed in high-throughput applications in the pharmaceutical industry.     

A microdevice with integrated liquid junction for facile peptide and protein analysis by capillary electrophoresis/electrospray mass spectrometry

A novel microfabricated device was implemented for facile coupling of capillary electrophoresis with mass spectrometry (CE/MS). The device was constructed from glass wafers using standard photolithographic/wet chemical etching methods. The design integrated (a) sample inlet ports, (b) the separation channel, (c) a liquid junction, and (d) a guiding channel for the insertion of the electrospray capillary, which was enclosed in a miniaturized subatmospheric electrospray chamber of an ion trap MS. The replaceable electrospray capillary was precisely aligned with the exit of the separation channel by a microfabricated guiding channel. No glue was necessary to seal the electrospray capillary. This design allowed simple and fast replacement of either the microdevice or the electrospray capillary. The performance of the device was tested for CE/MS of peptides, proteins, and protein tryptic digests. On-line tandem mass spectrometry was used for the structure identification of the protein digest products. High-efficiency/high-resolution separations could be obtained on a longer channel (11 cm on-chip) microdevice, and fast separations (under 50 s) were achieved with a short (4.5 cm on-chip) separation channel. In the experiments, both electrokinetic and pressure injections were used. The separation efficiency was comparable to that obtained from conventional capillary electrophoresis.     

A polymeric microfluidic chip for CE/MS determination of small molecules

A polymeric microfluidic chip made of Zeonor 1020 was fabricated using conventional embossing techniques to perform capillary electrophoresis for selected ion monitoring and selected reaction monitoring mass spectrometric detection of small molecules. A silicon master was microfabricated using photolithographic and dry etching processes. The microfluidic channel was embossed in the plastic from a silicon master. The embossed chip was thermally bonded with a Zeonor 1020 cover to form an enclosed channel. This channel (60-microm width, 20-microm depth, 2.0- and 3.5-cm length) provided capillary electrophoresis (CE) separation of polar small molecules without surface treatment of the polymer. A microsprayer coupled via a microliquid junction provided direct electrospray mass spectrometric detection of CE-separated components. An electric field of 0.5-2 kV/cm applied between the microsprayer and a separation buffer reservoir produced a separation of carnitine, acylcarnitine, and butylcarnitine with separation efficiencies ranging from 1,650 to 18,000 plates. Injection quantities of 0.2 nmol of these compounds produced a separation of the targeted polar small molecules without surface treatment of the polymer-abundant ion current signals and baseline separation of these compounds in less than 10 s. These results suggest the feasibility of polymeric chip-based devices for ion spray CE/MS applications.     

Toxin screening in phytoplankton: detection and quantitation using MALDI triple quadrupole mass spectrometry

The investigation of a MALDI triple quadrupole instrument for the analysis of spirolide toxins in phytoplankton samples is described in this study. A high-frequency (kHz) laser was employed for MALDI, generating a semicontinuous ion beam, thus taking advantage of the high duty cycle obtained in sensitive triple quadrupole MRM experiments. Initially, several experimental parameters such as type of organic matrix and concentration, solvent composition, and matrix-to-analyte ratio were optimized, and their impact on sensitivity and precision of the obtained ion currents for a reference spirolide, 13-desmethyl-C, was studied. In all quantitative experiments, excellent linearities in the concentration range between 0.01 and 1.75 microg/mL were obtained, with R2 values of 0.99 or higher. The average precision of the quantitative MALDI measurements was 7.4+/-2.4% RSD. No systematic errors were apparent with this method as shown by a direct comparison to an electrospray LC/MS/MS method. Most importantly, the MALDI technique was very fast; each sample spot was analyzed in less than 5 s as compared to several minutes with the electrospray assay. To demonstrate the potential of the MALDI triple quadrupole method, its application to quantitative analysis in several different phytoplankton samples was investigated, including crude extracts and samples from mass-triggered fractionation experiments. 13-Desmethyl spirolide C was successfully quantified in these complex samples at concentration levels from 0.05 to 90.4 microg/mL (prior to dilution to have samples fall within the dynamic range of the method) without extensive sample preparation steps. The versatility of the MALDI triple quadrupole method was also exhibited for the identification of unknown spirolide analogues. Through the use of dedicated linked scan functions such as precursor ion and neutral loss scans, several spirolide compounds were tentatively identified directly from the crude extract, without the usual time-consuming chromatographic preseparation steps. Moreover, high-quality CID spectra were obtained for low-abundant spirolides present in the phytoplankton samples.     

Fully automated chip-based mass spectrometry for complex carbohydrate system analysis

Carbohydrates represent a major class of biopolymers, which occur in nature either as oligosaccharides or glycoconjugates, in which the sugar moiety is linked to proteins or lipids. The significance of mass spectrometry for highly sensitive analysis of complex carbohydrates increased after the introduction of the electrospray ionization and matrix assisted laser desorption/ionization methods and the possibility of tandem MS for sequencing of single molecular species in complex mixtures. Rapid and sensitive characterization of carbohydrates in biological systems by automated nanoscale liquid delivery and chip-based electrospray interface techniques have not been developed so far. In this contribution, the implementation and optimization of a fully automated chip-based nanoelectrospray assembly (NanoMate system), operating in the negative ion mode, in combination with QTOF-tandem MS for mapping/sequencing and computer-assisted structure assignment for carbohydrate components in complex mixtures is presented.     

Structural characterization of protein tryptic peptides via liquid chromatography/mass spectrometry and collision-induced dissociation of their doubly charged molecular ions

The formation of multiply charged molecular ions via the field-assisted ion evaporation mechanism during electrospray ionization enables the use of an atmospheric pressure ionization quadrupole mass spectrometer system for characterizing biologically important peptides. The straightforward implementation of high-performance liquid chromatography (HPLC) into this new strategy to determine the molecular weight of tryptic peptides via the pneumatically assisted electrospray (ion spray) interface is presented. Examples utilizing both microbore (1.0 mm) and standard bore (4.6 mm) inside diameter columns are shown for the LC/MS molecular weight determination of tryptic peptides in methionyl-human growth hormone (met-hGH). Injected levels from 50 to 75 pmol of tryptic digest onto 1 mm i.d. HPLC columns provided full-scan LC/MS or LC/MS/MS results without postcolumn splitting of the effluent. When standard 4.6 mm i.d. HPLC columns were used, a 20:1 postcolumn split was utilized, which required from 1 to 5 nmol of injected tryptic digest for full-scan LC/MS or LC/MS/MS results. Collision-induced dissociation (CID) mass spectra resulting from either "infusion" or on-line LC/MS/MS analysis of the abundant doubly charged ions that predominate for tryptic peptides under electrospray conditions provided structurally useful sequence information for met-hGH and human hemoglobin tryptic digests. The slower mass spectrometer scan rate used during infusion of sample provides more accurate mass assignments than on-line LC/MS or LC/MS/MS, but the latter on-line experiments preclude ambiguities caused by matrix or component interferences. However, in some instances very weak CID product ions preclude complete tryptic peptide structural characterization based upon the CID data alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

LC/MS/MS determination of omapatrilat, a sulfhydryl-containing vasopeptidase inhibitor, and its sulfhydryl- and thioether-containing metabolites in human plasma

Omapatrilat, the most clinically advanced member of a new class of cardiovascular agents, vasopeptidase inhibitors, is under development at Bristol-Myers Squibb Pharmaceutical Research Institute for the treatment of hypertension and heart failure. An electrospray LC/MS/MS method has been developed and validated for the simultaneous determination of omapatrilat and its four metabolites in human plasma. Since omapatrilat and two of the metabolites are sulfhydryl-containing compounds, methyl acrylate was used to stabilize these compounds in human blood and plasma samples. Methyl acrylate reacted instantly with the sulfhydryl group to form a derivative that was stable in blood and plasma. Extraction of the analytes from plasma samples was achieved by semiautomated liquid-liquid extraction, where a robotic liquid handler performed the liquid-transferring steps. The mass spectrometer was operated in the negative ion selected-reaction-monitoring mode. The calibration curve ranges were 0.5-250 ng/mL for omapatrilat and one metabolite and 2.0-250 ng/mL for the other three metabolites.     

Chip-based P450 drug metabolism coupled to electrospray ionization-mass spectrometry detection

A chip-based P450 in vitro metabolism assay coupled with ESI-MS and ESI-MS/MS detection is described in this paper. The chips were made of a cyclic olefin polymer using a hot embossing process. The introduction of reagent solutions into the chip was carried out using fused-silica capillaries coupled to two syringes with the flow rate controlled by a syringe pump. Initial experiments described here employed a small commercial guard column in an off-chip format to desalt and concentrate the products of the enzymatic reaction prior to ESI-MS analysis. The system was used both to yield the Michaelis constant (K(m)) of the P450 biotransformation of imipramine into desipramine and to determine the IC50 value of a chemical inhibitor (tranylcypromine) for this CYP2C19-mediated reaction. The results demonstrated that the kinetics of the reaction inside the 4-microL volume within the channels of the cyclic olefin polymer chip provided results in agreement with those reported in the literature using conventional assays. The above reactions were carried out using human liver microsomes, and the metabolites were detected by ESI-MS showing the potential of the chip-based P450 reaction for metabolite screening studies as well as for P450 inhibition assays. A porous monolithic column was subsequently integrated into the chip to perform the reaction mixture cleanup process in an integrated fashion on the chip that is necessary for ESI-MS detection. The miniature monolithic SPE column was prepared in situ inside the chip via UV-initiated polymerization. The results obtained using the integrated system demonstrated the possibility of performing P450 enzymatic reactions in a microvolume reaction chamber coupled directly to ESI-MS detection and required less than 4 microg of HLM protein.     

Simple coupling of gas chromatography to electrospray ionization mass spectrometry

A simple method for direct coupling of gas chromatography (GC) with electrospray ionization mass spectrometry (ESI/MS) has been developed. The outlet of the GC capillary column was placed between the ESI needle and the atmospheric pressure ionization (API) source of a mass spectrometer. The ionization occurs via dissolution of neutral compounds into the charged ESI droplet followed by ion evaporation or via a gas-phase proton transfer reaction between a protonated solvent molecule and an analyte. The mass spectra of organic volatile compounds showed abundant protonated molecules with little fragmentation, being very similar to those produced by normal liquid ESI. The quantitative performance of the system was evaluated by determining the limit of detection (LOD), linearity ( r (2)), and repeatability (RSD). The GC-ESI/MS method was shown to be stable, providing high sensitivity and good quantitative performance.     

Development of a solid-phase extraction-HPLC/single quadrupole MS method for quantification of perfluorochemicals in whole blood

A method for the determination of perfluorooctanesulfonate (PFOS) and perfluorooctanoic acid (PFOA) simultaneously with 10 closely related perfluorochemicals (PFCs) in human whole blood was developed and validated. PFOS and PFOA are used in various applications, for example, as surfactants and plastic additives, and are subject to environmental and health research due to their persistence. The main part of the data on PFCs in human blood is from serum samples, analyzed mainly by ion pair extraction followed by high-performance liquid chromatography (HPLC) and negative electrospray (ESI) tandem mass spectrometry (MS/MS). The analytical method developed here is suitable for human whole blood and involves solid-phase extraction (SPE) and HPLC negative electrospray single quadrupole mass spectrometry (HPLC/ES-MS). A whole blood aliquot was treated with formic acid and extracted on a octadecyl (C18) SPE column. The PFCs were isolated with methanol, and quantification was performed using single quadrupole mass spectrometry and perfluoroheptanoic acid as internal standard. Validation was performed in the range 0.3-194 ng/mL with recovery between 64 and 112% and limit of detection in the 0.1-0.5 ng/mL range for 11 of the 12 PFCs studied. We applied this method to 20 whole blood samples collected in 1997-2000 from the Swedish population in the ages 24-72. Eleven of the 12 PFCs were detected, and they were quantitatively and qualitatively confirmed using triple quadrupole LC/MS/MS analysis. PFOS, perfluorooctanesulfonamide, perfluorohexanesulfonate, PFOA and perfluorononanoic acid were quantified in all samples. In addition, perfluorohexanoic acid, perfluorodecanoic acid, perfluorodecanesulfonate, perfluoroundecanoic acid, perfluorododecanoic acid, and perfluorotetradecanoic acid were detected in some samples. This study shows that SPE and single quadrupole MS can be applied for extraction and quantification of PFCs in human whole blood, resulting in selectivity and low detection limits.     

Molecular resolution and fragmentation of fulvic acid by electrospray ionization/multistage tandem mass spectrometry

Molecular weight distributions of fulvic acid from the Suwannee River, Georgia, were investigated by electrospray ionization/quadrupole mass spectrometry (ESI/ QMS), and fragmentation pathways of specific fulvic acid masses were investigated by electrospray ionization/ion trap multistage tandem mass spectrometry (ESI/MST/ MS). ESI/QMS studies of the free acid form of low molecular weight poly(carboxylic acid) standards in 75% methanol/25% water mobile phase found that negative ion detection gave the optimum generation of parent ions that can be used for molecular weight determinations. However, experiments with poly(acrylic acid) mixtures and specific high molecular weight standards found multiply charged negative ions that gave a low bias to molecular mass distributions. The number of negative charges on a molecule is dependent on the distance between charges. ESI/MST/MS of model compounds found characteristic water loss from alcohol dehydration and anhydride formation, as well as CO2 loss from decarboxylation, and CO loss from ester structures. Application of these fragmentation pathways to specific masses of fulvic acid isolated and fragmented by ESI/MST/MS is indicative of specific structures that can serve as a basis for future structural confirmation after these hypothesized structures are synthesized.     

Direct analysis in real time for reaction monitoring in drug discovery

Direct analysis in real time (DART) is a novel ionization technique that provides for the rapid ionization of small molecules under ambient conditions. In this study, several commercially available drugs as well as actual compounds from drug discovery research were examined by LC/UV/ESI-MS and DART interfaced to a quadrupole mass spectrometer. For most compounds, the molecular ions observed by ESI-MS were observed by DART/MS. DART/MS was also studied as a means to quickly monitor synthetic organic reactions and to obtain nearly instantaneous molecular weight confirmations of final products in drug discovery. For simple, synthetic organic transformations, the trends in the intensities of the mass spectral signals for the reactant and product obtained by DART/MS scaled closely with those of the diode array or the total ion chromatogram obtained by LC/UV/ESI-MS. In summary, DART is a new tool that complements electrospray ionization for the rapid ionization and subsequent mass spectral analysis of compounds in drug discovery.     

High-throughput screening and characterization of reactive metabolites using polarity switching of hybrid triple quadrupole linear ion trap mass spectrometry

A highly sensitive and efficient method has been developed for detection and characterization of glutathione (gamma-glutamyl-cysteinylglycine, GSH)-trapped reactive metabolites using a negative precursor ion (PI) as the survey scan to trigger the acquisition of positive enhanced product ion (EPI) spectra on a triple quadrupole linear ion trap mass spectrometer. The negative precursor ion scan step was carried out monitoring the anion at m/z 272, corresponding to deprotonated gamma-glutamyl-dehydroalanyl-glycine originating from the glutathionyl moiety. Because of the uniqueness and abundance of the anion at m/z 272, this single survey scan exhibited broad utility in the detection of unknown GSH conjugates. Further structural characterization was achieved by analyzing positive MS2 spectra that featured rich fragments without mass cutoff and were acquired in the same liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis. The effectiveness and reliability of this approach was evaluated using a number of model compounds in human liver microsomal incubations, including acetaminophen, clozapine, diclofenac, imipramine, meclofenamic acid, and ticlopidine. As a result, the PI-EPI approach revealed the presence of known adducts and, in many instances, identified additional conjugates that had not been reported previously. In comparison to the widely used neutral loss (NL) scanning analysis, this approach provided superior sensitivity and selectivity for different types of GSH conjugates. More importantly, the PI-EPI approach is suitable for high-throughput screening of reactive metabolites in the drug discovery process.     

Chip-based solid-phase extraction pretreatment for direct electrospray mass spectrometry analysis using an array of monolithic columns in a polymeric substrate

An array of eight porous monolithic columns, prepared in a Zeonor polymeric chip by UV-initiated polymerization of butyl methacrylate and ethylene dimethacrylate, was tested for solid-phase extraction (SPE) cleanup of biological samples prior to directly coupled electrospray mass spectrometry (ESI-MS). The chip, fabricated by hot embossing and thermal bonding, consists of eight parallel channels (10 mm long, 360 microm i.d.) connected via external fused-silica capillaries. The monomer mixture was aspirated simultaneously into the eight channels using a homemade vacuum manifold device and polymerized in parallel for 20 min under UV irradiation. The porous monolithic columns were then characterized by scanning electron microscopy and evaluated by ESI-MS applications with respect to sample capacity, recovery, reproducibility of peak area or peak height ratios, and linearity between peak height ratio and concentration using imipramine as a pharmaceutical test compound. The average sample capacity was estimated to be 0.30 microg with a relative standard deviation (RSD) of 26.5% for the eight monolithic columns on the same polymeric chip. For two chips prepared using the same monomer mixture, the difference in average sample capacity was 7.0%. The average recovery for the eight monolithic SPE columns on the same chip was 79.1% with an RSD of 7.9%. Using imipramine-d3 as an internal standard, the RSD of peak height ratios for the eight different columns was 2.0% for a standard solution containing 1 microg/mL imipramine. A linear calibration curve (R2 = 0.9995) was obtained for standard aqueous solutions of imipramine in the range from 0.025 to 10 microg/mL. To demonstrate the analytical potential of the chip-based SPE system, two different types of real-world samples including human urine sample and P450 drug metabolism incubation mixture were tested. Similar to standard aqueous solution, a linear correlation (R2 = 0.9995) was also found for human urine sample spiked with imipramine in the range of 0.025-10 microg/ mL. When aliquots of a human urine sample spiked with 1 microg/mL imipramine were loaded onto eight different monolithic columns, the RSD of peak height ratios was 3.8%. For a P450-imipramine incubation mixture, the formation of the N-demethylated metabolite (m/z 267.2) and the monohydroxylated metabolite (m/z 297.2) of imipramine was observed following chip-based monolithic SPE sample cleanup and preconcentration.     

On-line capillary electrophoresis/microelectrospray ionization-tandem mass spectrometry using an ion trap storage/time-of-flight mass spectrometer with SWIFT technology.

The development of a system capable of the speed required for on-line capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) of tryptic digests is described. The ion trap storage/reflectron time-of-flight (IT/reTOF) mass spectrometer is used as a nonscanning detector for rapid CE separation, where the peptides are ionized on-line using electrospray ionization (ESI). The ESI produced ions are stored in the ion trap and dc pulse injected into the reTOF-MS at a rate sufficient to maintain the separation achieved by CE. Using methodology generated by software and hardware developed in our lab, we can produce SWIFT (Stored Waveform Inverse Fourier Transform) ion isolation and TICKLE activation/fragmentation voltage waveforms to generate MS/MS at a rate as high as 10 Hz so that the MS/MS spectra can be optimized on even a 1-2 s eluting peak. In CE separations performed on tryptic digests of dogfish myelin basic protein (MBP) where eluting peaks 4-8 s wide are observed, it is demonstrated that an acquisition rate of 4 Hz provides > 20 spectra/peak and is more than sufficient to provide optimized MS/MS spectra of each of the eluting peaks in the electropherogram. The detailed structural analysis of dogfish MBP including several posttranslational modifications using CE-MS and CE-MS/MS is demonstrated using this method with < 10 fmol of material consumed.     

MS for identification of single nucleotide polymorphisms and MS/MS for discrimination of isomeric PCR products

ESI (electrospray ionization) MS and tandem mass spectrometry (MS/MS) were used for the analysis of single nucleotide polymorphisms (SNPs) and more complex genetic variations. Double-stranded (ds) PCR products were studied. PCR products of the proline [5'-x(G17)-x(C38)x-3'] and arginine variants [(5'-x(Gl7)-x(G38)x-3'] of the p53 gene are distinguished by an SNP (cytosine or guanine) and were discriminated using both quadrupole and quadrupole ion trap MS analysis. A 69 bp arginine mutant PCR product [5'-x(C17)-x(G38)x-3'] with a negating switch has the same mass as the proline variant but was readily distinguishable on ion trap MS/MS analysis; fragments containing the mutation site, but not the polymorphism, were identified. The 69 bp PCR products were restriction-enzyme-digested, to create 43 bp fragments. ESI quadrupole ion trap MS/MS analysis of the 43 bp product-ion spectra readily demonstrated both polymorphism and negating switch sites. MS and MS/MS are powerful and complementary techniques for analysis of DNA. MS can readily distinguish SNPs but MS/MS is required to differentiate isomeric PCR products (same nucleotide composition but different sequence).     

Electrospray Ion Chromatography-Tandem Mass Spectrometry of Bromate at Sub-ppb Levels in Water

An electrospray ion chromatography-tandem mass spectrometry (IC-MS/MS) method has been developed for the analysis of bromate ions in water. This IC-MS/MS method improves the limit of detection of bromate ions by a factor of 10. The method consists of solid phase extraction with an ion exchange column and elution of the analyte with water/methanol ammonium sulfate eluent on-line with a negative ion electrospray mass spectrometry detection. SPE requires sample pretreatment to remove any major ions that displace bromate, consisting of eliminating SO(4)(2)(-), Cl(-), and HCO(3)(-) ions respectively with barium-form, silver-form, and acid (H(+)-form) exchange resins. The methanolic sulfate eluent permits IC-MS coupling via an electrospray interface. BrO(3)(-) was selected in the first quadrupole (Q1) at two m/z values, 127 and 129, according to the isotope contributions of (79)Br and (81)Br. After fragmentation in the collision cell (second quadrupole, Q2), the third quadrupole (Q3) analyzes the product ions as (M - O)(-), (M - 2O)(-), and (M - 3O)(-). Among the six recordable transitions, four were selected, the other two yielding high background. A lowered resolution raised sensitivity by a factor of up to 3. The limit of quantitation of this method was 0.1 μg/L.     

Evaluation of a four-channel multiplexed electrospray triple quadrupole mass spectrometer for the simultaneous validation of LC/MS/MS methods in four different preclinical matrixes

A four-channel multiplexed electrospray interface on a triple quadrupole mass spectrometer was evaluated for the simultaneous validation of LC/MS/MS methods for the quantitation of loratadine and its metabolite, descarboethoxyloratadine, in four different biological matrixes. The assays were performed in rat, rabbit, mouse, and dog plasma from 1 to 1000 ng/mL using 96-well solid-phase extraction for sample preparation. The limit of quantitation of 1 ng/mL corresponded to 5.56 pg of each analyte injected on-column. For the drug, quality control samples (n = 6 at four concentrations) had precision ranging from 0.967 to 16.0% and accuracy ranging from -8.44 to 10.5% across all four species. For the metabolite, the precision ranged from 0.684 to 11.0% and the accuracy was between 6.36 and -9.06%. Intersprayer cross talk for the multiplexed electrospray ion source was evaluated as a function of analyte concentration and was less than 0.08% at concentrations as high as 1000 ng/mL. These results demonstrate the feasibility of using parallel analysis to reduce the time required for method validation and to increase sample throughput in drug development studies.     

A comparison of the quantitative methods for the analysis of the platinum-containing anticancer drug [cis-[amminedichloro(2-methylpyridine)]platinum(II)] (ZD0473) by HPLC coupled to either a triple quadrupole mass spectrometer or an inductively coupled plasma mass spectrometer

The use of high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC-ICPMS) as means for the quantitative determination of ZD0473, a platinum anticancer drug, and its related biologically active "aqua" compounds in biofluid samples is described. The performance of the resulting HPLC-ICPMS method was compared with that of a conventional HPLC-triple quadrupole mass spectrometer-based (HPLC-MS/MS) system for properties such as limit of detection, linearity, and reproducibility using spiked samples. The methods were then applied to the determination of plasma ultrafitrate concentrations of ZD0473 in dog plasma samples obtained following intravenous and oral administration at 0.5 and 6 mg/kg, respectively. These experiments showed that both methods were capable of providing accurate and precise results but that the HPLC-ICPMS method had advantages of extended linear range and superior sensitivity, providing a limit of quantification of 0.1 ng/mL for ZD0473, as compared to 5 ng/mL using the current HPLC-MS/MS method. In addition, by using a single combined HPLC-ICPMS/MS/MS system, it was possible to determine the relative MS/MS response of the aqua compounds for the first time.