Biomedical subjects

Jack Henion

Publications and source records attributed to Jack Henion.

8 recordsLinked to original sources

PubMed2005

Determination of pharmaceutical compounds in aqueous dimethyl sulfoxide by electrospray ionization mass spectrometry.

Standard solutions of reserpine, dextromethorphan, imipramine and amitriptyline in dimethyl sulfoxide (DMSO), DMSO containing 0.1% formic acid, and DMSO/H(2)O (1:1, v/v) containing 0.1% formic acid were analyzed by positive electrospray ionization mass spectrometry (ESI-MS). A triple-quadrupole mass spectrometer equipped with a TurboIonspray (TIS) interface was used for the ESI-MS analyses. The samples dissolved in the respective DMSO solution were infused directly into the mass spectrometer at 10 microL/min using an infusion pump. The positive Q1 full-scan (m/z 50-650) mass spectrum of DMSO (MW = 78) showed three main peaks at m/z 79, 101 and 179, corresponding to the protonated molecule [DMSO+H](+), and the sodiated adducts [DMSO+Na](+) and [2DMSO+Na](+), respectively. The ESI of the compounds in DMSO and DMSO containing 0.1% formic acid was promoted by using the TIS gas (GS2) combined with the nebulizer gas (GS1), and TIS source temperature set to 250 degrees C. In contrast, samples dissolved in DMSO/H(2)O (1:1, v/v) containing 0.1% formic were sprayed at a lower temperature (100 degrees C) using only the nebulizer gas. The TIS voltage (V) was optimized in order to establish the lowest voltage necessary to achieve optimum ESI of each pharmaceutical compound with the voltage maintained below the onset potential required to produce a corona discharge at the TIS probe (sprayer). Detection limits of 10 ng/mL were achieved for reserpine, dextromethorphan, imipramine and amitriptyline in each solvent composition.

Dimethyl Sulfoxide↗

PubMed2004

High-throughput liquid chromatography-tandem mass spectrometry determination of bupropion and its metabolites in human, mouse and rat plasma using a monolithic column.

In the present work, a high-throughput LC/MS/MS method using a Chromolith RP-18 (50 mm x 4.6 mm) monolithic column was developed and partially validated for the determination of bupropion (BUP), an anti-depressant drug, and its metabolites, hydroxybupropion and threo-hydrobupropion (TB), in human, mouse, and rat plasma. A modern integrated liquid chromatograph and an LC/MS/MS system with a TurboIonSpray (TIS) interface were used for the positive electrospray selected reaction monitoring (SRM) LC/MS analyses. Spiked control plasma calibration standards and quality control (QC) samples were extracted by semi-automated 96-well liquid-liquid extraction (LLE) using ethyl acetate. A mobile phase consisting of 8mM ammonium acetate-acetonitrile (55:45, v/v) delivered isocratically at 5 ml/min, and split post-column to 2 ml/min directed to the TIS, provided the optimum conditions for the chromatographic separation of bupropion and its metabolites within 23s. The isotope-labeled D(6)-bupropion and D(6)-hydroxybupropion were used as internal standards. The method was linear over a concentration range of 0.25-200 ng/ml (bupropion and threo-hydrobupropion), and 1.25-1000 ng/ml (hydroxybupropion). The intra- and inter-day assay accuracy and precision were within 15% for all analytes in each of the biological matrices. The monolithic column performance as a function of column backpressure, peak asymmetry, and retention time reproducibility was adequately maintained over 864 extracted plasma injections.

Animals↗

PubMed2003

A device for automated direct sampling and quantitation from solid-phase sorbent extraction cards by electrospray tandem mass spectrometry.

A new solid-phase extraction (SPE) device in the 96-well format (SPE Card) has been employed for automated off-line sample preparation of low-volume urine samples. On-line automated analyte elution via SPE and direct quantitation by micro ion spray mass spectrometry is reported. This sample preparation device has the format of a microtiter plate and is molded in a plastic frame which houses 96 separate sandwiched 3M Empore sorbents (0.5-mm-thickness, 8-microm particles) covered on both sides by a microfiber support material. Ninety-six discrete SPE zones, each 7 mm in diameter, are imbedded into the sheet in the conventional 9-mm pitch (spacing) of a 96-well microtiter plate. In this study one-quarter of an SPE Card (24 individual zones) was used merely as a convenience. After automated off-line interference elution of applied human urine from 24 samples, a section of SPE Card is mounted vertically on a computer-controlled X, Y, Z positioner in front of a micro ion spray direct sampling tube equipped with a beveled tip. The beveled tip of this needle robotically penetrates each SPE elution zone (sorbent disk) or stationary phase in a serial fashion. The eluted analytes are sequentially transferred directly to a microelectrosprayer to obtain tandem mass spectrometric (MS/MS) analysis. This strategy precludes any HPLC separation and the associated method development. The quantitative determination of Ritalin (methylphenidate) from fortified human urine samples is demonstrated. A trideuterated internal standard of methylphenidate was used to obtain ion current response ratios between the parent drug and the internal standard. Human control urine samples fortified from 6.6 to 3300 ng/mL (normal therapeutic levels have been determined in other studies to be between 50 and 100 ng/mL urine) were analyzed and a linear calibration curve was obtained with a correlation coefficient of 0.9999, where the precision of the quality control (QC) samples ranged from 9.6% at the 24 ng/mL QC level to 1.2% at the 3000 ng/mL QC level, and the accuracy for the four levels of QC samples ranged from 98.1% to 100.3%. The QC samples were prepared at four concentrations which included 24, 240, 1200, and 3000 ng/mL, respectively. The run time per sample in this work was 1.5 min not including the sample preparation time.

Automation↗

PubMed2003

High-throughput selected reaction monitoring liquid chromatography-mass spectrometry determination of methylphenidate and its major metabolite, ritalinic acid, in rat plasma employing monolithic columns.

This work presents a high-throughput selected reaction monitoring (SRM) LC-MS method for the determination of methylphenidate (MPH), a central nervous stimulant, and its de-esterified metabolite, ritalinic acid (RA) in rat plasma samples. A separation of these two compounds was achieved in 15 s by employing a 3.5-ml/min flow-rate, a porous monolithic column and a TurboIonSpray source compatible with relatively high flow-rates. In addition, a relatively fast autosampler and a new data acquisition system resulted in a time lag of less than 17 s between consecutive injections. Overall, 768 protein-precipitated rat plasma samples (eight 96-well plates) containing both MPH and RA were analyzed within 3 h and 45 min. The partial method validation described in this report included an assessment of linearity, intra and inter-assay precision and accuracy, and method robustness. Deuterated internal standards for the target compounds, d(3)-MPH and d(5)-RA, were employed. The calibration curves ranged from 0.1 to 50 ng/ml for MPH and from 0.5 to 50 ng/ml for RA. The limit of quantification (LOQ) for MPH and RA was 0.1 and 0.5 ng/ml, respectively. For both analytes, the intra- and inter-assay precision (relative standard deviation, % C.V.) and accuracy (relative error) did not exceed 15% for the quality control samples (QCs) QC1, QC2 or QC3 (0.3, 1.5 and 40 ng/ml for MPH and 0.15, 15 and 40 ng/ml for RA) for either analyte and did not exceed 20% at the lower limit of quantitation (LOQ) level. No carry-over from the autosampler was detected. The retention times remained constant throughout the experiment. Baseline resolution of MPH and RA was consistently observed throughout the plates analyzed. The described method demonstrates the feasibility for employing monolithic HPLC columns to effect rapid bioanalytical SRM LC-MS analysis of representative biological samples.

Animals↗

PubMed2003

A fully automated nanoelectrospray tandem mass spectrometric method for analysis of Caco-2 samples.

Caco-2 cells offer a means to rapidly screen permeability of drug candidates, allowing pharmaceutical companies to eliminate candidates unable to cross the intestinal barrier early in the discovery process. This screening process is typically performed by conventional liquid chromatography/tandem mass spectrometry (LC/MS/MS), which can require time-consuming method development. An alternative to LC/MS/MS, automated nanoelectrospray tandem mass spectrometry (nanoESI-MS/MS), is introduced. This novel approach requires an off-line ZipTip desalting step followed by automated nanoESI-MS/MS, using the NanoMate 100 and ESI Chip. In addition to reduced method development time, automated nanoESI-MS/MS also offers no carry-over between samples, low sample consumption, and ease-of-use as compared with conventional pulled-capillary nanoelectrospray. Furthermore, the infusion system described has the potential to be high-throughput. A comparison of Caco-2 samples analyzed both by LC/MS/MS and by automated nanoESI-MS/MS is presented. The permeability and recovery data of the two compounds analyzed in this study obtained from conventional LC/MS/MS and by automated nanoESI-MS/MS were in excellent agreement.

Algorithms↗

PubMed2003

Quantitation of midazolam in human plasma by automated chip-based infusion nanoelectrospray tandem mass spectrometry.

An automated chip-based infusion nanoelectrospray ionization (nanoESI) platform was used to demonstrate reproducible quantitation of drug molecules from biological matrices. Three sample preparation strategies were explored including protein precipitation of plasma with acetonitrile, de-salting of the plasma, and a combination of protein precipitation with subsequent de-salting of the dried and reconstituted extract. The best results were obtained when fortified human plasma samples containing midazolam were precipitated with acetonitrile containing alprazolam as the internal standard (IS). The supernatant was concentrated to dryness, reconstituted in aqueous acid, and de-salted by automated reversed-phase solid-phase extraction (SPE) prior to infusion nanoESI-MS/MS. Analyses employed a triple quadrupole mass spectrometer operated in selected reaction monitoring (SRM) mode. Each sample was infused for approximately 10 s and the resulting ion current profiles were integrated. Area ratios were used for regression analysis of standard samples (1.5-500 ng/mL). Quality control samples (3, 250, and 400 ng/mL) in five replicates from three different analysis days demonstrated intra-assay precision (< or =16%), inter-assay precision (< or =5%), and overall accuracy (+/-9% deviation). Infusion reproducibility of the assay was established by analyzing extracts after storage for 24 h at ambient temperature. Control plasma samples from six different sources probed the potential utility of this technique for the analysis of clinical samples. At the lower limit of quantitation (LLQ), variability and mean overall accuracy were < or =13% CV and +/-3% deviation, respectively, while at the upper limit of quantitation (ULQ) variability and mean overall accuracy were < or =9% CV and +/-9% deviation, respectively. Inter-chip variability was established by determining standard sample extracts across five different chips (< or =12% CV). Throughput for the assay was 55 s per sample, although this time may be shortened to 40 s per sample with recent improvements in the automated nanoESI system. No contamination or carryover was observed using this promising automated nanoESI-MS/MS platform.

Automation↗

PubMed2002

Atmospheric pressure photoionization liquid chromatographic-mass spectrometric determination of idoxifene and its metabolites in human plasma.

This paper describes a comparison between atmospheric pressure chemical ionization (APCI) and the recently introduced atmospheric pressure photoionization (APPI) interface for the LC-MS determination of idoxifene and its major metabolite, SB245419 (SB19), in human plasma. The results indicate that analyte response in APPI is highly dependent on the solvent composition, especially to water in the mobile phase. Other parameters investigated are the mobile phase flow-rate, the chemical noise, and signal suppression by matrix interferences. APPI appears to be six to eight times more sensitive than APCI for idoxifene and its SB245419 metabolite; the response for the SB245420 metabolite is considerably better than for APCI conditions, but still not sufficient for trace level pharmacokinetic determinations in human plasma. The LOQ for the parent drug and its major metabolite were 10 and 25 ng/ml, respectively, in human plasma. From post-column infusion experiments we conclude that there is little difference in matrix suppression between APCI and APPI. From these studies we suggest APPI may be an additional tool in pharmaceutical LC-MS applications.

Atmospheric Pressure↗

PubMed2002

Surface-alkylated polystyrene monolithic columns for peptide analysis in capillary liquid chromatography-electrospray ionization mass spectrometry.

Macroporous poly(styrene-divinylbenzene) (PS-DVB) monoliths were prepared by in situ polymerization in PEEK, fused silica, or stainless steel tubing having an inner diameter of 75 or 125 microm. A process is described for subsequent alkylation of the flow-contacting surfaces of the monoliths. The process treats all the surfaces including through-pore surfaces of the rigid macroporous monolith with a solution containing a dissolved Friedel-Crafts catalyst, an alkyl halide (1-chlorooctadecane), and an organic solvent. This process produces an improved reversed-phase liquid chromatographic separation of peptides compared to an unmodified monolithic PS-DVB column. The surface octadecylation is not necessary for a reversed-phase separation of proteins since both unmodified and modified columns provide comparable results. Tryptic protein digests, standard proteins, and standard peptides were used to evaluate the monolithic columns by employing electrospray mass spectrometry detection. Potential applications in proteomics studies by mass spectrometry, which use the alkylated monolithic column engaged onto the nanofabricated electrospray ionization chip, are also discussed.

Alkylation↗