Dennis W. Hill

HPLC Photodiode Array UV Detection for Toxicological Drug Analysis

Abstract An HPLC/UV system for the analysis of drugs and toxic compounds is presented. Retention data for 157 compounds in an acid HPLC system and 144 compounds in a basic HPLC system are listed. UV spectral data for 25 compounds from each system are listed. The usefulness of a computer search routine, utilizing UV and HPLC retention data, is discussed.

Quantitative HPLC Analysis of Plasma Amino Acids as Orthophthaldialdehyde/Ethanethiol Derivatives

Abstract A reverse phase high performance liquid chromatographic method for the analysis of plasma amino acids is described. The method employs pre-column derivatization with o-phthaldialdehyde using ethanethiol as the reducing agent. The analysis shows good linearity and reproducibility. An average overall difference of 12% was seen for results obtained by the HPLC method versus those obtained with an amino acid analyzer. The chromatographic parameters of buffer concentration and column temperature were also examined.

CE50: Quantifying collision induced dissociation energy for small molecule characterization and identification

Survival yield analysis is routinely used in mass spectroscopy as a tool for assessing precursor ion stability and internal energy. Because ion internal energy and decomposition reaction rates are dependent on chemical structure, we reasoned that survival yield curves should be compound-specific and therefore useful for chemical identification. In this study, a quantitative approach for analyzing the correlation between survival yield and collision energy was developed and validated. This method is based on determining the collision energy (CE) at which the survival yield is 50% (CE50) and, further, provides slope and intercept values for each survival yield curve. In initial experiments using a defined set of homologous compounds, we found that CE50 values were easily determined, quantitative, highly reproducible, and could discriminate between structural and even positional isomers. Further analysis demonstrated that CE50 values were independent of cone potential and orthogonal to compound mass. Experimentally determined CE50 values for a diverse set of 54 compounds were correlated to Molconn molecular structure descriptors. The resulting model yielded a statistically significant linear correlation between experimental and calculated CE50 values and identified several structural characteristics related to precursor ion stability and fragmentation mechanism. Thus, the CE50 is a promising method for compound identification and discrimination.

In silico enzymatic synthesis of a 400,000 compound biochemical database for nontargeted metabolomics.

Current methods of structure identification in mass-spectrometry-based nontargeted metabolomics rely on matching experimentally determined features of an unknown compound to those of candidate compounds contained in biochemical databases. A major limitation of this approach is the relatively small number of compounds currently included in these databases. If the correct structure is not present in a database, it cannot be identified, and if it cannot be identified, it cannot be included in a database. Thus, there is an urgent need to augment metabolomics databases with rationally designed biochemical structures using alternative means. Here we present the In Vivo/In Silico Metabolites Database (IIMDB), a database of in silico enzymatically synthesized metabolites, to partially address this problem. The database, which is available at http://metabolomics.pharm.uconn.edu/iimdb/, includes ~23,000 known compounds (mammalian metabolites, drugs, secondary plant metabolites, and glycerophospholipids) collected from existing biochemical databases plus more than 400,000 computationally generated human phase-I and phase-II metabolites of these known compounds. IIMDB features a user-friendly web interface and a programmer-friendly RESTful web service. Ninety-five percent of the computationally generated metabolites in IIMDB were not found in any existing database. However, 21,640 were identical to compounds already listed in PubChem, HMDB, KEGG, or HumanCyc. Furthermore, the vast majority of these in silico metabolites were scored as biological using BioSM, a software program that identifies biochemical structures in chemical structure space. These results suggest that in silico biochemical synthesis represents a viable approach for significantly augmenting biochemical databases for nontargeted metabolomics applications.

Improved GC/MS Analysis of Opiates with Use of Oxime-TMS Derivatives

An improved gas chromatographic/mass spectrometric (GC/MS) assay is described for the quantitation of codeine and morphine as trimethylsyl (TMS) derivatives. The TMS derivatization of ketone-containing opiates results in the formation of multiple derivatives. Some of these products have retention times close to those of codeine-TMS and morphine-TMS. When the keto-opiates are present in samples assayed for codeine and morphine in urine, they can interfere with the quantitation of these commonly targeted opiates. The assay was improved with the addition of a pre-BSTFA derivatization step, whereby hydroxylamine was used to convert the keto-opiates into the corresponding oxime derivative. These derivatives were then reacted with BSTFA to form the TMS ethers and TMS oxime derivatives. The oxime step enabled production of single derivatives for hydrocodone and hydromorphone. In addition, the retention times for the oxime-TMS derivatives were increased so that they no longer elute near the targeted drugs of codeine and morphine. The addition of the oxime step does not affect the sylation of codeine and morphine, and the accuracy and precision of this assay were unaffected.

High Performance Liquid Chromatographic Separation and Fluorescent Measurement of Taurine, a Key Amino Acid

Abstract A rapid, sensitive method for the analysis of taurine in oyster hemolymph (blood) has been developed. Highly fluorescent, substituted isoindoles formed by the reaction of taurine and other amino acids with o-phthaldialdehyde/ethanethiol reagent were separated on a reverse phase, high performance liquid chromatographic column. It was necessary to carefully control the concentration of the sodium ion in the phosphate buffer in order to maintain an adequate resolution of both taurine and tyrosine from β-alanine and arginine. Calibration plots of the fluorescent taurine derivative were linear over 2.5 orders of magnitude with a limit of detection of 0.10 nanomoles per ml of oyster hemolymph.

The Anticarcinogenic Conjugated Fatty Acid, 9c, 11t-18:2, in Human Milk: Confirmation of Its Presence

The concentration of the anticarcinogenic fatty acid, 9c, lIt-1 8:2, in human milk was determined by gas-liquid chromatography (GLC). The mean concentration of 20 samples from 5 women taken at 1, 7, 14, and 21 days was: 0.18% + 0.02; range, 0.14-0.28%. Identity was confirmed by GLC-mass spectrometry (MS). Conjugated isomers other than 9c, 1 lt-18:2 were not detected. The amounts were not changed by supplementation of the maternal diet with fish oil beginning on day 1 after the milk sample was taken.

Evaluation of Alkyl Bonded Silica and Solvent Phase Modifiers for the Efficient Elution of Basic Drugs on HPLC

Abstract Nine representative drugs were used to evaluate the effects of alkyl bonded stationary phases containing type A and type B silica and the effects of an amine modifier on the efficiency of high performance liquid chromatographic elution of basic and acidic drugs. The theoretical plate count and asymmetry factor of the eluted peaks were compared to that of acetophenone as a reference to the maximal efficiency of each system evaluated. ZorbaxR C8 was used as the stationary phase prepared from type A silica and Zorbax RXR was used as the stationary phase prepared from the type B silica. The theoretical plate count and asymmetry factor of acetophenone was observed to be the same on both columns when analyzed in an acidic aqueous/acetonitrile mobile phase. An improvement in the efficiency and peak shape of the amine containing compounds was observed using the Zorbax RXR stationary phase as compared to the efficiency and peak shape of these compounds on the ZorbaxR C8 stationary phase. Interestingly, th...

Desmosterol in human milk

Milk samples were collected from mothers at 2,6, 12 and 16 weeks postpartum. Desmosterol was found to be present in all the milk samples. Identification of desmosterol was based on retention times with two gas liquid chromatography (GLC) columns and verified by GC-m ass spectrometry. The concentration of desmosterol in breast milk increased significantly (P<0.5) from 0.6 mg/100 ml at 2 weeks to 1.3 mg/100 ml at 16 weeks postpartum. Desmosterol was not significantly correlated with total lipid, total cholesterol or free cholesterol in the milk.

Definitive Identification of an Exceptionally High Methanol Concentration in an Intoxication of a Surviving Infant: Methanol Metabolism by First-Order Elimination Kinetics

Intoxication by methanol was identified in a five-week-old infant suffering from moderate metabolic acidosis. The initial serum methanol at admission was 1148 mg/dL as measured by gas chromatography. The osmolal gap and formic acid concentrations were consistent with methanol intoxication. The child was treated with folic acid and a continuous ethanol infusion and survived without any apparent permanent problems. Because expected toxic symptoms did not develop in this case, and the methanol concentrations were at levels that might be deemed to be incompatible with life, blood and urine samples were assayed by a specific enzymatic assay, and by gas chromatography/mass spectrometry (GC/MS). Positive results definitively confirmed the presence of methanol. In contrast to previous reports, the elimination of methanol in this case appeared to following first-order kinetics. If hepatic ADH activity is low in neonates and young infants, another enzyme system such as catalase may be involved to explain this data. The lack of formic acid accumulation may have been due to folic acid therapy.