Rick C. Steenwyk

Nanostructure Initiator Mass Spectrometry: Tissue Imaging and Direct Biofluid Analysis

Nanostructure initiator mass spectrometry (NIMS) is a recently introduced matrix-free desorption/ionization platform that requires minimal sample preparation. Its application to xenobiotics and endogenous metabolites in tissues is demonstrated, where clozapine and N-desmethylclozapine were observed from mouse and rat brain sections. It has also been applied to direct biofluid analysis where ketamine and norketamine were observed from plasma and urine. Detection of xenobiotics from biofluids was made even more effective using a novel NIMS on-surface extraction method taking advantage of the hydrophobic nature of the initiator. Linear response and limit of detection were also evaluated for xenobiotics such as methamphetamine, codeine, alprazolam, and morphine, revealing that NIMS can be used for quantitative analysis. Overall, our results demonstrate the capacity of NIMS to perform sensitive, simple, and rapid analyses from highly complex biological tissues and fluids.

Detection of Carbohydrates and Steroids by Cation-Enhanced Nanostructure-Initiator Mass Spectrometry (NIMS) for Biofluid Analysis and Tissue Imaging

Nanostructure-initiator mass spectrometry (NIMS) is a highly sensitive, matrix-free technique that is well suited for biofluid analysis and imaging of biological tissues. Here we provide a new technical variation of NIMS to analyze carbohydrates and steroids, molecules that are challenging to detect with traditional mass spectrometric approaches. Analysis of carbohydrates and steroids was accomplished by spray depositing NaCl or AgNO(3) on the NIMS porous silicon surface to provide a uniform environment rich with cationization agents prior to desorption of the fluorinated polymer initiator. Laser desorption/ionization of the ion-coated NIMS surface allowed for Na(+) cationization of carbohydrates and Ag(+) cationization of steroids. The reliability of the approach is quantitatively demonstrated with a calibration curve over the physiological range of glucose and cholesterol concentrations in human serum (1-200 microM). Additionally, we illustrate the sensitivity of the method by showing its ability to detect carbohydrates and steroids down to the 800-amol and 100-fmol levels, respectively. The technique developed is well suited for tissue imaging of biologically significant metabolites such as sucrose and cholesterol. To highlight its applicability, we used cation-enhanced NIMS to image the distribution of sucrose in a Gerbera jamesonii flower stem and the distribution of cholesterol in a mouse brain. The flower stem and brain sections were placed directly on the ion-coated NIMS surface without further preparation and analyzed directly. The overall results reported underscore the potential of NIMS to analyze and image chemically diverse compounds that have been traditionally challenging to observe with mass spectrometry-based techniques.

Comparison of fused-core and conventional particle size columns by LC–MS/MS and UV: Application to pharmacokinetic study

The chromatographic performance of fused-core (superficially porous) HPLC packing materials was compared with conventional fully porous particle materials for LC-MS/MS analysis of two pharmaceuticals in rat plasma. Two commercially available antidepressants, imipramine and desipramine, were assayed using a conventional analytical C(18) column (5 microm, 2.0 mm x 30 mm) and a fused-core C(18) column (2.7 microm, 2.1 mm x 30 mm). Retention time, column efficiency, pressure drop, resolution, and loading capacity were compared under the same operating conditions. The fused-core column demonstrated reduced assay time by 34% and 2-3-fold increased efficiency (N). Loading capacity up to 25 microl of extract injected on column showed no peak distortion. The registered back-pressure from a flow rate of 1.0 ml/min did not exceed 3400 psi making it compatible with standard HPLC equipment (typically rated to 6000 psi). Two mobile phases were examined, and morpholine as an organic base modifier yielded a 2-5-fold increase in S/N near the limit of detection over triethylamine. The 2.7 microm fused-core column was applied to the analysis of imipramine and desipramine in extracted, protein precipitated rat plasma by LC-MS/MS. The calibration curves were linear in the concentration range of 0.5-1000 ng/ml for both imipramine and desipramine. Intra-run precisions (%CV) and accuracies (%bias) were within +/-7.8% and +/-7.3% at three QC levels and within 14.7% and 14.4% at the LOQ level for both analytes. Following a single method qualification run, the method was applied to the quantitation of pharmacokinetic study samples after oral administration of imipramine to male rats.

Ultra sensitive quantitation of endogenous oxytocin in rat and human plasma using a two-dimensional liquid chromatography-tandem mass spectrometry assay.

Oxytocin (OT) is a neuropeptide with an extremely low endogenous level (low pg/ml) in human plasma. It is very challenging to develop a highly sensitive assay to measure endogenous OT, including radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Electrospray ionization (ESI) liquid chromatography-tandem mass spectrometry (LC-MS/MS) can provide high-throughput and selective methods for quantification of peptides in biological samples. A novel and highly sensitive two-dimensional LC-MS/MS (2D-LC-MS/MS) assay combining solid-phase extraction (SPE) has been developed and validated for the determination of endogenous OT in both human and rat plasma. The lower limit of quantification (LLOQ) was 1.00 pg/ml for human and 50.0 pg/ml for rat. Human plasma diluted with water (1:6, v/v) was successfully optimized as a surrogate matrix for human to prepare standard curves without endogenous interference. The extraction efficiency and absolute recovery were above 65.8% using the HLB SPE procedure, and matrix effects were lower than 12%. The method was validated in the range of 1.00-250 pg/ml for human plasma and 50.0-10,000 pg/ml for rat plasma with precision less than 12.7% and accuracy less than 7%.

Ultra sensitive measurement of endogenous epinephrine and norepinephrine in human plasma by semi-automated SPE-LC-MS/MS.

Measurement of endogenous epinephrine (E) and norepinephrine (NE) in human plasma is very challenging due to lower endogenous concentrations as compared with animal plasma. An LC-MS/MS in combination with alumina-based SPE and derivatization procedure was validated for the measurement of E and NE in human plasma with acceptable intra-day and inter-day accuracy and precision. Sample was extracted with semi-automated alumina 96-well solid phase extraction (SPE) cartridge. The resulting eluent was dried and derivatized using d4-acetaldehyde. The analytes were separated on a monolithic C(18) column. Extraction efficiencies were >66% for E and NE. The lower limit of quantitation (LLOQ) was 5.00 pg/mL for E and 20.0 pg/mL for NE.

Development and validation of a sample stabilization strategy and a UPLC–MS/MS method for the simultaneous quantitation of acetylcholine (ACh), histamine (HA), and its metabolites in rat cerebrospinal fluid (CSF)

A UPLC-MS/MS assay was developed and validated for simultaneous quantification of acetylcholine (ACh), histamine (HA), tele-methylhistamine (t-mHA), and tele-methylimidazolacetic acid (t-MIAA) in rat cerebrospinal fluid (CSF). The biological stability of ACh in rat CSF was investigated. Following fit-for-purpose validation, the method was applied to monitor the drug-induced changes in ACh, HA, t-mHA, and t-MIAA in rat CSF following administration of donepezil or prucalopride. The quantitative method utilizes hydrophilic interaction chromatography (HILIC) Core-Shell HPLC column technology and a UPLC system to achieve separation with detection by positive ESI LC-MS/MS. This UPLC-MS/MS method does not require extraction or derivatization, utilizes a stable isotopically labeled internal standard (IS) for each analyte, and allows for rapid throughput with a 4 min run time. Without an acetylcholinesterase (AChE) inhibitor present, ACh was found to have 1.9±0.4 min in vitro half life in rat CSF. Stability studies and processing modification, including the use of AChE inhibitor eserine, extended this half life to more than 60 min. The UPLC-MS/MS method, including stabilization procedure, was validated over a linear concentration range of 0.025-5 ng/mL for ACh and 0.05-10 ng/mL for HA, t-mHA, and t-MIAA. The intra-run precision and accuracy for all analytes were 1.9-12.3% CV and -10.2 to 9.4% RE, respectively, while inter-run precision and accuracy were 4.0-16.0% CV and -5.3 to 13.4% RE, respectively. By using this developed and validated method, donepezil caused increases in ACh levels at 0.5, 1, 2, and 4h post dose as compared to the corresponding vehicle group, while prucalopride produced approximately 1.6- and 3.1-fold increases in the concentrations of ACh and t-mHA at 1h post dose, respectively, compared to the vehicle control. Overall, this methodology enables investigations into the use of CSF ACh and HA as biomarkers in the study of these neurotransmitter systems and related drug discovery efforts.

Strategies for quantitation of endogenous adenine nucleotides in human plasma using novel ion-pair hydrophilic interaction chromatography coupled with tandem mass spectrometry

We present here a novel and highly sensitive ion-pair hydrophilic interaction chromatography-tandem mass spectrometry (IP-HILIC-MS/MS) method for quantitation of highly polar acid metabolites like adenine nucleotides. A mobile phase based on diethylamine (DEA) and hexafluoro-2-isopropanol (HFIP) and an aminopropyl (NH2) column were applied for a novel chromatographic separation for the determination of AMP, ADP and ATP in biological matrices. This novel IP-HILIC mechanism could be hypothesized by the ion-pairing reagent (DEA) in the mobile phase forming neutral and hydrophilic complexes with the analytes of polar organic acids. The IP-HILIC-MS/MS assay for adenine nucleotides was successfully validated with satisfactory linearity, sensitivity, accuracy, reproducibility and matrix effects. The lower limit of quantitation (LLOQ) at 2.00ng/mL obtained for ATP showed a least 10-fold higher sensitivity than previous LC-MS/MS assays except nano-LC-MS/MS assay. In summary, this novel IP-HILIC-MS/MS assay provides a sensitive method for nucleotides bioanalysis and shows great potential to determine a number of organic acids in biological matrices.

Simultaneous and high-throughput quantitation of urinary tetranor PGDM and tetranor PGEM by online SPE-LC-MS/MS as inflammatory biomarkers

Quantitation of urinary tetranor PGDM or tetranor PGEM (tPGDM and tPGEM) in the past was performed separately using off-line SPE LC-MS/MS methods. The manual SPE procedure is generally time-consuming and cost-ineffective. In addition, simultaneous quantitation of tPGDM and tPGEM is favorable yet very challenging because of the similar chemical structures and identical MRM transitions. This work describes the development and validation of a high-throughput online SPE-LC-MS/MS method, allowing simultaneous and high-throughput measurement of tPGDM and tPGEM in human urine. The reportable range of the assay was 0.2-40 ng/ml for tPGDM and 0.5-100 ng/ml for tPGEM. Intra- and inter-assay precision and accuracy determined using quality control samples were all within acceptable ranges (% CV and % Bias < 15%). Tetranor PGDM was stable under all tested conditions while tPGEM was stable at 4 °C and after three F/T cycles but not stable at room temperature for 24 h (recovery below 80%). The assay was applied to measure urinary tPGDM and tPGEM among healthy volunteers, smokers and COPD patients. Significantly higher urinary levels of both tPGDM and tPGEM were observed in COPD patients than those of non-smoking healthy volunteers. These results demonstrated that the high-throughput online SPE-LC-MS/MS assay provides sensitive, reproducible and accurate measurement of urinary tPGDM and tPGEM as biomarkers for assessing inflammatory diseases such as COPD.

Inhibition of in vitro lipid peroxidation by 21-aminosteroids : evidence for differential mechanisms

In a previous report (Ryan and Petry, Arch Biochem Biophys 300: 699-704, 1993), the effects of two 21-aminosteroids (U-74500A and U-74006F) on the oxidation and reduction of iron in a buffer/organic solvent system were investigated. In those studies, U-74500A was found to be an efficient iron reductant and potential iron chelator, whereas U-74006F had little effect on iron redox chemistry. As an extension of those studies, we now report the effects of U-74006F and U-74500A on lipid peroxidation in systems that are dependent upon iron oxidation/reduction. In liposomes, U-74500A inhibited ADP:Fe(II)-dependent lipid peroxidation in a concentration-dependent manner, whereas U-74006F was minimally effective in this system. The mechanism of U-74500A-dependent inhibition probably involved interactions with iron, as iron oxidation was inhibited in the presence of this compound. No effects on iron oxidation were observed in the presence of U-74006F. Addition of Ferrozine to liposomal incubation mixtures indicated that at least two iron pools were present in samples containing U-74500A, one immediately bound by Ferrozine, and another that was bound more slowly. Furthermore, ADP:Fe(III)/ascorbate-dependent lipid peroxidation was blocked completely by U-74500A, presumably by formation of a redox inert complex upon reduction of the iron. U-74500A partially protected ADP:Fe(II) from oxidation by H2O2 and lipid hydroperoxides, indicating that the U-74500A:iron complex was stable in the presence of biologically relevant oxidants. U-74006F did not markedly affect iron oxidation or reduction when incorporated into phospholipid liposomes. In microsomal lipid peroxidation systems containing ADP:Fe(III) and NADPH, both U-74500A and U-74006F inhibited lipid peroxidation. U-74006F-dependent inhibition of microsomal lipid peroxidation was dependent on both NADPH and Fe(III). Further, it was enhanced when U-74006F was allowed to preincubate in this system prior to iron addition. Preincubation of U-74006F with microsomes, NADPH, and ADP:Fe(III) produced several metabolites detectable by HPLC. These results suggest that U-74500A inhibits lipid peroxidation by directly affecting iron redox chemistry, whereas U-74006F-mediated inhibition is enhanced by preincubation with a metabolically competent microsomal system.

Quantification of urinary PGEm, 6-keto PGF1α and 2,3-dinor-6-keto PGF1α by UFLC–MS/MS before and after exercise

Eicosanoids play an important role in the evaluation of pro-inflammatory responses and in the safety and toxicity of novel therapeutic agents. This work describes a high-throughput UFLCMS/MS method for the analysis of three urinary prostanoid biomarkers of pro-inflammatory responses, tetranor PGEm, 6-keto PGF(1alpha) and 2,3-dinor-6-keto PFG(1alpha). Nine male volunteers of various age and fitness level participated in this study. Six provided pre- and post-exercise samples and three provided intraday samples. Tetranor PGEm and 6-keto PGF(1alpha) increased significantly in patients after exercise (p<0.017 and p<0.029). In individual patient sets, tetranor PGEm levels increased from 1.5- to 6-fold pre- vs. post-exercise, levels of 6-keto PGF(1alpha) increased more dramatically from 2- to 55-fold pre- vs. post-exercise. The prostanoid 2,3-dinor-6-keto PGF(1alpha) remained unchanged post-exercise. Data was normalized to urinary creatinine concentration, which increased approximately 40% post-exercise.