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D E Moody

Publications and source records attributed to D E Moody.

At least 37 records · Page 2Linked to original sources
PubMed1999

Long-term stability of abused drugs and antiabuse chemotherapeutical agents stored at -20 degrees C.

Stability is an important consideration in the use of specimens for accurate determination of analyte concentrations. To determine the long-term stability for analytes routinely analyzed by mass spectrometry in this laboratory, quality-control (QC) results were plotted versus time. The time required for the initial concentration to reach a specified level of deviation (i.e., 15%) was then determined from the slopes. QCs were prepared at 1-3 concentrations in drug-free matrix and stored at approximately -20 degrees C; urines were fortified with 1% sodium fluoride; plasmas (except for cocaines) were prepared with heparin. For cocaine and metabolites, the plasma was either fortified with 2% sodium fluoride or with 2% sodium fluoride and 1 mg% physostigmine after adjustment of the plasma pH to 6.0. In urine, amphetamine, methamphetamine, codeine, morphine, benzoylecgonine (BZE), and 11-nor-9-carboxy-delta9-tetrahydrocannabinol (THCA) slopes did not exceed a 15% deviation before 852 days. Cocaine, however, reached a 15% reduction at 165 days. When cocaine and BZE were prepared in plasma with just 2% sodium fluoride, negative slopes reached 15% deviation within 154 and 111 days, respectively. Further fortification with physostigmide and adjustment of the pH extended this time frame significantly. Delta9-Tetrahydrocannabinol (THC) and THCA in plasma had negative slopes that deviated by 15% just prior to one year. l-Alpha-acetylmethadol (LAAM), methadone, and their N-demethylated metabolites in urine did not have any negative slopes exceeding 15% before 686 days. Several of the compounds had positive slopes. Those for 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine reached the 15% mark within 98 days. Those for LAAM, norLAAM, and dinorLAAM were concentration dependent. The 25-ng/mL controls reached 15% at 158-216 days. The 700-ng/mL controls reached 15% at 784-1340 days. In plasma, only naltrexone and buprenorphine displayed negative slopes at all three concentrations, reaching the 15% mark as early as 576 and 272 days, respectively. LAAM, norLAAM, dinorLAAM, ibogaine, 6-beta-naltrexone, risperidone, and 9-OH-risperidone did not exceed a 15% deviation before 416 days. To attempt to validate this method, two sets of clinical plasma samples that had been analyzed for buprenorphine were reanalyzed 644 and 869 days after the initial analyses. Those reanalyzed after 644 days were not statistically different from initial analyses, whereas those stored for 869 days were statistically different (p < 0.05). As the average time to reach 15% deviation for the three concentrations of buprenorphine QCs was 782 days, this suggests that extrapolation of QC results gathered over time may provide a reliable method to estimate long-term stability limits for drugs stored under the same conditions as the QC samples.

Cryopreservation↗
PubMed1998

Effects of cannabinoids on preimplantation mouse embryo development and implantation are mediated by brain-type cannabinoid receptors.

We examined the relative importance of G (Gi) protein-coupled brain-type (CB1-R) and spleen-type (CB2-R) cannabinoid receptors in preimplantation embryo development using agonists and antagonists specific to CB1-R and CB2-R. The results establish that endogenous cannabinoid ligands, anandamide and sn-2 arachidonoylglycerol, arrest embryo development in vitro, and this effect is reversed by CB1-R antagonists SR141716A or AM 251, but not by SR144528, a CB2-R antagonist. A CB2-R selective agonist AM 663 failed to affect embryo development. These results suggest that cannabinoid effects on embryo development are mediated by CB1-R. We also observed that delta9-tetrahydrocannabinol ([-]THC) infused in the presence of cytochrome P450 inhibitors interfered with blastocyst implantation. This adverse effect was reversed by coinfusion of SR141716A. The less active stereoisomer (+)THC plus the inhibitors failed to affect implantation. Analysis of tissue levels demonstrated that uterine accumulation of (-)THC occurred when it was infused in the presence of the P450 inhibitors. These results demonstrate that the uterus and perhaps the embryo have the cytochrome P450 enzymes to metabolize (-)THC and neutralize its adverse effects on implantation. Collectively, the present study demonstrates that cannabinoid effects on embryo development and implantation are mediated by embryonic and/or uterine CB1-R, but not CB2-R.

Animals↗
PubMed1997

Variability in metabolic rate, feed intake and fatness among selection and inbred lines of mice.

Mouse populations differing in metabolic rate have been developed through selection for high (MH) and low (ML) heat loss (HLOSS), along with randomly selected controls (MC). Objectives of this study were to (a) compare MH, ML and MC lines for HLOSS and correlated traits of feed intake, body composition and organ weights; (b) compare three widely used inbred mouse lines with MH, ML and MC for the same traits; and (c) investigate potential genotype by diet interaction resulting from feeding diets differing in fat percentage. Heat loss (kcal/day) of MH and ML mice differed by 37% of the mean and remained significant (33%) when HLOSS was expressed on a fat-free mass basis. MH mice consumed more energy than ML with a greater difference in mice fed high-fat compared with standard diets (27% vs 13.9%). Despite greater energy consumption, MH mice were leaner than ML with a difference in total body fat percentage of 40%. The greatest difference in HLOSS between selection and inbred lines was between MH and C57BL/6J (BL), which differed by 26.3%. MH and BL mice also differed in energy intake (15.5%). Body composition of BL mice was similar to MH when fed a standard diet, but similar to ML when fed a high-fat diet. Crosses between MH and ML or between MH and BL would be useful to investigate the genetic regulation of, and identify quantitative trait loci influencing HLOSS, energy intake and body composition. Feeding of a high-fat diet may allow diet-specific loci influencing body composition to be identified in MH and BL lines.

Adipose Tissue, Brown↗
PubMed1997

Determination of buprenorphine in human plasma by gas chromatography-positive ion chemical ionization mass spectrometry and liquid chromatography-tandem mass spectrometry.

Buprenorphine is used for the management of pain and has been advocated for the treatment of opioid addiction. Therapeutic doses result in low plasma concentrations of buprenorphine. In order to assess the safety and efficacy of buprenorphine, sensitive analytical methods are needed. Until recently, gas chromatography-positive ion chemical ionization mass spectrometry (GC-PCI-MS) offered the most sensitive method to selectively quantitate buprenorphine. We have developed and validated a sensitive liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) method for buprenorphine. The method is described and compared with a GC-PCI-MS method validated in this laboratory. One-milliliter aliquots of plasma are required for the LC-ESI-MS-MS method and 2-mL aliquots for the GC-PCI-MS method. Buprenorphine-d4 is used as internal standard for both methods. Derivatization with pentafluoropropionic acid anhydride is used for the GC-PCI-MS method, in which the derivatized protonated molecular ions after loss of water are monitored at m/z 596 and 600. For LC-ESI-MS-MS, the parent protonated molecule ions are monitored at m/z 468 and 472. A single-step extraction of basic plasma with n-butyl chloride provided recoveries of 70-87%. Although a limit of quantitation (LOQ) of 0.1 ng/mL could be established for LC-ESI-MS-MS, we could only achieve an LOQ of 0.5 ng/mL with the GC-PCI-MS assay. The GC-PCI-MS method has a linear range of 0.5 to 40 ng/mL (mean r2 = 0.998, n = 7). For quality control samples at 1.0, 2.5, and 12.5 ng/mL, the intra- and interassay coefficients of variation (CV) did not exceed 14%, and percent of targets were within 16%. The LC-ESI-MS-MS method had a linear range of 0.1 to 10 ng/mL (mean r2 = 0.999, n = 7). For quality control samples at 0.25, 2.5 and 7.5 ng/mL, the intra- and interassay CVs did not exceed 4%, and percent of targets were within 12%. Stability studies demonstrated buprenorphine was stable for up to 24 h, 125 days, and 55 days when stored at room temperature, 4 degrees C, and -20 degrees C, respectively. The utility of the lower LOQ was demonstrated in 40 plasma samples collected up to 96 h after a sublingual dose of buprenorphine; 10 were quantitatable using GC-PCI-MS and 38 using LC-ESI-MS-MS.

Administration, Sublingual↗
PubMed1997

Feasibility of the grandprogeny design for quantitative trait loci (QTL) detection in purebred beef cattle.

The grandprogeny design (GPD) was developed for dairy cattle to use existing pedigreed populations for quantitative trait locus (QTL) detection. Marker genotypes of grandsires and sons are determined, and trait phenotypic data from grandprogeny are analyzed. The objective of this study was to investigate the potential application of GPD in purebred beef cattle populations. Pedigree structures of Angus (n = 123,319), Hereford (n = 107,778), Brangus (n = 14,449), and Gelbvieh (n = 8,114) sire evaluation reports were analyzed to identify potentially useful families. Power of QTL detection was calculated for a range of QTL effects (.1 to .5 SD) and two Type I error rates (.01 and .001). Reasonable power (> .75) could be achieved using GPD in Angus and Hereford for QTL having moderate effects (.3 SD) on weaning weight and large effects (.4 to .5 SD) on birth, yearling, and maternal weaning weights by genotyping 500 animals. Existing Gelbvieh and Brangus families useful for GPD were limited, and reasonable power could be expected only for QTL having large effects on weaning or birth weights. Although family structures suitable for GPD exist in purebred beef populations, large amounts of genotyping would be required to achieve reasonable power, and only QTL having moderate to large effects could be expected to be identified.

Aging↗
PubMed1997

The involvement of cytochrome P450 3A4 in the N-demethylation of L-alpha-acetylmethadol (LAAM), norLAAM, and methadone.

The N-demethylation of LAAM, norLAAM, and methadone has been investigated in human liver microsomes and microsomes containing cDNA-expressed human P450s. Gas chromatography/mass spectrometry methods allowed detection of norLAAM and dinorLAAM formation from LAAM, dinorLAAM formation from norLAAM, and EDDP and EMDP formation from methadone. The rates of N-demethylation varied 4- to 7-fold in microsomes from four different donors with activities for LAAM and norLAAM consistently greater (5- to 14-fold) than for methadone. The N-demethylation of LAAM, norLAAM, and methadone were significantly inhibited by ketoconazole. IC50s could be determined for ketoconazole inhibition of LAAM and norLAAM N-demethylation of 1.6 and 1.1 microM, respectively. The ability of ketoconazole to reduce methadone N-demethylation below 40% varied in regard to liver donor. No other P450-selective inhibitors reduced the average activities more than 43%. cDNA-expressed P450 3A4 N-demethylated LAAM, norLAAM, and methadone at greater rates than the other cDNA-expressed P450s studied (1A2, 2C9, 2D6, or 2E1). P450 3A N-demethylation of LAAM, norLAAM, and methadone exceeded the next most active P450, respectively, by at least 2.5, 9.6, and 13.4 times when expressed per milligram protein and by 18.2, 6.0, and 6.1 times when expressed per nanomole P450. These results suggest that P450 3A4 is the primary site of N-demethylation of LAAM, norLAAM, and methadone in human liver. Although other enzymes may also be capable of N-demethylating these compounds, identification of specific enzymes, except P450 3A4, has yet to be established. Knowledge of these enzymatic pathways is essential for assessment of the impact of metabolic drug-drug interactions on therapeutic success and/or adverse events.

Cytochrome P-450 CYP3A↗
PubMed1996

Correlations of the induction of microsomal epoxide hydrolase activity with phase II drug conjugating enzyme activities in rat liver.

Within the selective induction of phase II enzymes following treatment with dipyridyls or N-heterocyclic analogs of phenanthrene, strong correlations (r > or = 0.70) are observed between the increase of microsomal epoxide hydrolase (mEH) activity and UDP-glucuronosyltransferase (UGT) activities towards 4-nitrophenol, 1-naphthol and morphine. The present study investigates whether this correlation is maintained with inducing agents known to also increase phase I enzyme activities. Rats were treated with beta-naphthoflavone, isosafrole, phenobarbital, ethanol, dexamethasone and clofibric acid regimens in which P450 isozyme induction could be confirmed. Comparisons between the responses of mEH, UGT and glutathione S-transferase (GST) activities were made. mEH activity was increased by beta-naphthoflavone, isosafrole, phenobarbital and clofibric acid. The elevation in mEH activity by these agents showed modest but significant correlations with GST activities toward all the substrates monitored (r values range between 0.49 and 0.65) and a strong correlation with UGT activity towards only one substrate, morphine (r = 0.70). This study suggests that induction of mEH activity correlates with the increases in select phase II enzyme activities whether it is accompanied by P450 induction or not.

Animals↗
PubMed1996

Determination of methadone and its N-demethylation metabolites in biological specimens by GC-PICI-MS.

Methadone is often invoked for detoxification and maintenance of the opioid addict. We have developed and validated a sensitive and specific method for the analysis of methadone and its metabolites, 2-ethylidene-1,5-dimethyl-3, 3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenylpyrroline (EMDP), in human plasma, urine, and liver microsomes. This assay uses a solid-phase extraction. Separation and analysis of the analytes are performed by capillary gas chromatography-positive ion chemical ionization-mass spectrometry. The protonated molecules (MH+) are monitored at m/z 264 and 267 for EMDP-d0 and -d3, m/z 278 and 281 for EDDP-d0 and -d3, and m/z 310 and 313 for methadone-d0 and -d3. The recovery of methadone and its metabolites exceeded 85% in the different matrices analyzed. Linear standard curves in plasma and in urine were obtained over the concentration range of 10-600 ng/mL (coefficients of determination: methadone, > or = 0.995; EMDP, > or = 0.994; and EDDP, > or = 0.996). With plasma and urine fortified at 25, 100, and 300 ng/mL, the assay was precise (intra-assay coefficients of variation [CVs], 2-12%; interassay CVs, 1-15%) and accurate (intra-assay percent of target, 85-107; interassay percent of target, 88-105) for all three analytes. Stability studies indicated that methadone and its metabolites are stable at room temperature in plasma and in urine for at least 1 week and in liver microsomes for at least 24 h. This method has now been shown to be useful for quantitation of methadone, EDDP, and EMDP in human urine and plasma and is also useful for quantitation of the amount of EDDP produced in human liver microsomes incubated with methadone. It provides an accurate and precise analytical tool for further studies on the metabolism of methadone.

Ammonia↗
PubMed1996

Characterization of DNA polymorphisms in three populations of hereford cattle and their associations with growth and maternal EPD in line 1 herefords.

Three populations of Hereford cattle differing in inbreeding levels and genetic potential for growth were genotyped for seven DNA polymorphisms. The populations were compared to determine differences in allele frequency and genetic variation. Significant differences in allele frequency among the populations were found at six of the seven polymorphisms genotyped, and average genetic variation differed as expected when inbreeding levels were considered. Effects of several polymorphisms on growth and maternal EPD were evaluated for one population (Miles City Line 1 Herefords) using regression analysis. Substitution of a B allele for an A allele of the kappa-casein polymorphism accounted for significant decreases in direct birth weight and maternal 180-d gain from birth to weaning EPD explaining 15% and 8%, respectively, of EPD variability. Several other significant effects accounting for small portions of EPD variability were also detected.

Alleles↗
PubMed1995

A radiometric TLC assay of liver microsomal dextromethorphan O-demethylation.

A simple and sensitive assay for in vitro analysis of dextromethorphan O-demethylation, a marker for P450 2D deficiency in both humans (2D6) and rats (2D1), has been devised. Commercially available [N-methyl-3H]-dextromethorphan was used to develop a radiometric TLC assay for dextromethorphan O-demethylation. Hexane-triethylamine efficiently extracted dextromethorphan and metabolites from rat liver microsomes, and a solvent system of cyclohexane-toluene-diethylamine (65:15:20, v/v/v) provided sufficient separation (approximately 2 cm) between the two radioactive bands, dextromethorphan and dextrorphan, and no interference from the unlabeled N-demethylation products, 3-methoxymorphinan and 3-hydroxymorphinan. The recovery of dextrorphan from TLC plates increases with microsomal protein and incubation time. An eight-fold decrease in activity was noted in female Dark Agouti relative to the male Sprague-Dawley rats, respective models for poor and extensive P450 2D metabolizers. The assay, even with an approximately 100-fold dilution of radiolabeled substrate, had an approximate limit of detection of 100 pmol. Within- and between-run imprecision was 12.4% and 7.2%, respectively. The radiometric TLC assay for dextromethorphan O-demethylation was sensitive and easy, and used readily available equipment.

Animals↗
PubMed1995

Immunoassay detection of benzodiazepines and benzodiazepine metabolites in blood.

We evaluated the capability of commercially available benzodiazepine immunoassays from four vendors (Abuscreen radioimmunoassay [RIA-a] from Roche Diagnostic Systems and serum RIA-d from Diagnostic Products Corporation; X-systems serum and urine fluorescence polarization immunoassays [FPIA] from Abbott Laboratories; and EMIT TOX serum and EMIT urine enzyme immunoassays [EIA] from Syva) to detect 22 benzodiazepines and 16 benzodiazepine metabolites in blood samples. Drug-free bovine blood was fortified with the different compounds at concentrations relevant to blood analysis: 7, 30, and 70 ng/mL. One-milliliter samples were extracted with n-butyl chloride, dried, and reconstituted with 0.3 ml. of buffer. Results from the two urine-specific kits (EIA-u and FPIA-u) produced either equivalent or reduced cross-reactivity compared with their serum kit counterparts; these results are presented but not discussed further. Among the 38 compounds, the following were detected by all four assays at concentrations of 70 ng/mL or less: alprazolam, alpha-hydroxy-alprazolam, norclobazam, diazepam, estazolam, flunitrazepam, norflunitrazepam, miclazolam, 4-hydroxy-midazolam, nitrazepam, noradinazolam, oxazepam, prazepam, 3-hydroxy-prazepam, temazepam, and triazolam. 7-Amino-clonazepam, 7-acetamido-clonazepam, 3-hydroxy-7-acetamidoclonazepam, haloxazepam, loprazolam, 7-amino-nitrazepam, 7-acetamido-nitrazepam, and oxazolam were not detected by any of the assays at concentrations up to 70 ng/mL. A number of benzodiazepines or their metabolites were detectable by some but not all of the six assays: bromazepam, chlordiazepoxide, demoxepam, and 4-hydroxy-alprazolam (both RIAs and EIA); adinazolam (RIA-a, EIA, and FPIA); norchlordiazepoxide (both RIAs); clonazepam (RIA-a and EIA); bentazepam, clotiaze-pam, and mexazolam (EIA and FPIA); 7-amino-flunitrazepam and quazepam (EIA); and alpha-hydroxy-triazolam and medazepam (RIA-a).

Animals↗
PubMed1995

A gas chromatographic-positive ion chemical ionization-mass spectrometric method for the determination of I-alpha-acetylmethadol (LAAM), norLAAM, and dinorLAAM in plasma, urine, and tissue.

l-alpha-Acetylmethadol (LAAM) is approved as a substitute for methadone for the treatment of opiate addiction. Analytical methods are needed to quantitate LAAM and its two psychoactive metabolites, norLAAM and dinorLAAM, to support pharmacokinetic and other studies. We developed a gas chromatographic-positive ion chemical ionization-mass spectrometric method for these analyses. The method uses 0.5 mL urine or 1.0 mL plasma or tissue homogenate, deuterated (d3) isotopomers as internal standards, methanolic denaturation of protein (for plasma and tissue), and extraction of the buffered sample with n-butyl chloride. For tissue homogenates, an acidic back extraction is included. norLAAM and dinorLAAM were derivatized with trifluoroacetic anhydride. Chromatographic separation of LAAM and derivatized norLAAM and dinorLAAM is achieved with a 5% phenyl methylsilicone capillary column. Positive ion chemical ionization detection using methane-ammonia as the reagent gas produces abundant protonated ions (MH+) for LAAM (m/z 354) and LAAM-d3 (m/z 357) and ammonia adduct ions (MNH4+) for the derivatized norLAAM (m/z 453), norLAAM-d3 (m/z 45 6), dinorLAAM (m/z 439), and dinorLAAM-d3 (m/z 442). The linear range of the calibration curves were matrix dependent: 5-300 ng/mL for plasma, 10-1000 ng/mL for urine, and 10-600 ng/g for tissue homogenates. The low calibrator was the validated limit of quantitation for that matrix. The method is precise and accurate with percent coefficients of variation and percent of targets within 13%. The method was applied to the analysis of human urine and plasma samples; rat plasma, liver, and brain samples; and human liver microsomes following incubation with LAAM.

Acetic Anhydrides↗
PubMed1995

Analysis of cocaine and its metabolites from biological specimens using solid-phase extraction and positive ion chemical ionization mass spectrometry.

An accurate and reliable gas chromatographic-mass spectrometric method was developed to analyze tissue, whole blood, plasma, and urine samples for cocaine (COC) and its major metabolites. COC, benzoylecgonine (BZE), and ecgonine methyl ester (EME) were isolated from the biological matrix using solid-phase extraction, and the tert-butyldimethylsilyl derivatives of BZE, EME, and their deuterium-labeled internal standards were formed. Separation of the compounds was performed by capillary chromatography, and analysis was performed by positive ion chemical ionization mass spectrometry using methane and ammonia as the reagent gases. The tert-butyldimethylsilyl derivatives of BZE and EME were stable and produced mass spectral ions with higher mass-to-charge ratios than trimethylsilyl derivatives. Recovery of COC and its metabolites exceeded 80% at all three concentrations tested. Linearity of the method was established from 2.5 to 2000 microg/L. Intra-assay precision had a coefficient of variation (CV) of less than 9% for all analytes when tested at 10, 25, 100, and 200 microg/L. Interassay precision also had a CV of less than 9% for COC, BZE, and EME at 25 and 100 microg/L. At 200 microg/L, %CVs for COC, BZE, and EME were 11.5, 12.0, and 12.7, respectively. In addition to the analysis of COC, BZE, and EME, the method was used to quantitate cocaethylene and to identify norcocaine.

Calibration↗
PubMed1995

Determination of ibogaine and 12-hydroxy-ibogamine in plasma by gas chromatography-positive ion chemical ionization-mass spectrometry.

Ibogaine, an indolamine derivative, is currently being investigated as a potential agent in the treatment of stimulant and opiate addiction. We developed a rapid, sensitive, and specific method for the analysis of ibogaine and its putative active metabolite, 12-hydroxy-ibogamine (12-OH-ibogamine). This assay employs a one-step basic extraction with n-butyl chloride-acetonitrile (4:1), followed by derivatization of the metabolite using N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide. The derivatized extracts were analyzed by capillary gas chromatography-positive ion chemical ionization-mass spectrometry. The ions monitored were at m/z 311, 314, and 411, which correspond to the protonated molecules (MH+) for ibogaine, ibogaine-d3, and 12-OH-ibogamine.tert-butyldimethylsilyl, respectively. Linear standard curves were obtained over the concentration range of 1 0-1 000 ng/mL (average r2, 0.995 for ibogaine and 0.992 for 12-OH-ibogamine; n = 3). Limits of quantitation were 10 ng/mL. The interrun and intrarun coefficients of variation for the assay of ibogaine at 25, 100, and 300 ng/mL ranged from 2.9 to 8.8%. We also established the extraction and chromatographic conditions to monitor the 12-hydroxylated metabolite. A suitable internal standard was not yet obtained so the method could only provide semiquantitative information for 12-OH-ibogamine. Chemical stability studies of these analytes indicated that ibogaine and 12-OH-ibogamine were stable in a human plasma matrix at room temperature for a period of at least 1 week.

Acetamides↗