Identification of quantitative trait loci associated with egg quality, egg production, and body weight in an F2 resource population of chickens.

Egg production and egg quality are complex sex-limited traits that may benefit from the implementation of marker-assisted selection. The primary objective of the current study was to identify quantitative trait loci (QTL) associated with egg traits, egg production, and body weight in a chicken resource population. Layer (White Leghorn hens) and broiler (Cobb-Cobb roosters) lines were crossed to generate an F2 population of 508 hens over seven hatches. Phenotypes for 29 traits (weekly body weight from hatch to 6 weeks, egg traits including egg, albumen, yolk, and shell weight, shell thickness, shell puncture score, percentage of shell, and egg shell colour at 35 and 55 weeks of age, as well as egg production between 16 and 55 weeks of age) were measured in hens of the resource population. Genotypes of 120 microsatellite markers on 28 autosomal groups were determined, and interval mapping was conducted to identify putative QTL. Eleven QTL tests representing two regions on chromosomes 2 and 4 surpassed the 5% genome-wise significance threshold. These QTL influenced egg colour, egg and albumen weight, percent shell, body weight, and egg production. The chromosome 4 QTL region is consistent with multiple QTL studies that define chromosome 4 as a critical region significantly associated with a variety of traits across multiple resource populations. An additional 64 QTL tests surpassed the 5% chromosome-wise significance threshold.

Ankyrin repeat and SOCS box protein 15 regulates protein synthesis in skeletal muscle.

Ankyrin repeat and SOCS box protein 15 (ASB15) is an Asb family member expressed predominantly in skeletal muscle. We have previously reported that ASB15 mRNA abundance decreases after administration of beta-adrenergic receptor agonists. Because beta-adrenergic receptor agonists are known to stimulate muscle hypertrophy, the objective of this study was to determine whether ASB15 regulates cellular processes that contribute to muscle growth. Stable myoblast C2C12 cells expressing full-length ASB15 (ASB15-FL) and ASB15 lacking the ankyrin repeat (ASB15-Ank) or SOCS box (ASB15-SOCS) motifs were evaluated for changes in proliferation, differentiation, protein synthesis, and protein degradation. Expression of ASB15-FL caused a delay in differentiation, followed by an increase in protein synthesis of approximately 34% (P<0.05). A consistent effect of ASB15 overexpression was observed in vivo, where ectopic expression of ASB15 increased skeletal muscle fiber area (P<0.0001) after 9 days. Expression of ASB15-SOCS altered differentiation of myoblasts, resulting in detachment of cells from culture plates. Expression of ASB15-Ank increased protein degradation by 84 h of differentiation (P<0.05), and in vivo ectopic expression of an ASB15 construct lacking both the ankyrin repeat and SOCS box motifs decreased skeletal muscle fiber area (P<0.0001). Together, these results suggest ASB15 participates in the regulation of protein turnover and muscle cell development by stimulating protein synthesis and regulating differentiation of muscle cells. This is the first study to demonstrate a role for an Asb family member in skeletal muscle growth.

Validation of dual-energy X-ray absorptiometry in live White Leghorns.

Dual energy x-ray absorptiometry (DEXA) was evaluated for use as a noninvasive tool to monitor skeletal integrity in live laying hens. The objectives of the current study were 1) to validate the use of DEXA in evaluating bone integrity in live birds as compared with excised bones under a normal nutritional regimen as well as in hens fed varying levels of dietary Ca and 2) to correlate densitometric scans with other bone strength criteria and egg traits. Densitometric scans were conducted on the tibia and humerus of live hens at 10-wk intervals from 17 to 67 wk of age. After each scan, bones were excised from euthanized hens to measure breaking strength characteristics and bone ash (experiment 1). Similar measurements were collected at 38, 48, and 58 wk of age from hens fed hypercalcemic (5.4%), control (3.6%), and hypocalcemic (1.8%) diets from 32 to 58 wk of age (experiment 2). The bone mineral density (BMD) and bone mineral content (BMC) between live and excised bone scans were highly correlated (r = 0.85 and 0.92, respectively, P < 0.0001, experiment 1). Densitometric scans of live birds were positively correlated with bone breaking force and bone ash (r = 0.68 and 0.73, respectively, P < 0.001) with little to no correlation with shell traits. In experiment 2, the excised tibial scan had lower BMD and BMC than the live bird (P < 0.01), whereas no difference was detected in densitometric scans of the humerus. The live and excised BMD and BMC of the tibia (r = 0.87 and 0.82, respectively, P < 0.001) and humerus (r = 0.94 and 0.93, respectively, P < 0.001) were highly correlated. Due to the high correlations between live and excised bone scans and the significant correlations of live scans to more traditional invasive bone measurement tests such as bone breaking force and bone ash, we concluded that DEXA is a useful noninvasive tool for evaluating skeletal integrity in live birds.

Effects of ovulatory and egg laying cycle on bone mineral density and content of live White Leghorns as assessed by dual-energy X-ray absorptiometry.

Dual-energy X-ray absorptiometry has been validated in our laboratory as a noninvasive tool to assess skeletal integrity in live birds. The first objective of the current study was to determine if there were detectable changes in bone mineral density (BMD) and bone mineral content (BMC) while an egg was being formed in the oviduct. Implications from this experiment would define the time of day scans should be conducted for future experiments. Densitometric scans were conducted on the tibia and humerus of live hens undergoing active egg formation when hens were 0, 5, 15, and 20 h postoviposition at 24, 30, and 40 wk of age. No detectable changes in either the BMD or BMC of the tibia and humerus were observed as the egg was being formed in the reproductive tract at any age measured. These results suggest that densitometric scans may be conducted on bones in live birds at any time during the day, irrespective of the stage of egg formation. The second objective was to monitor the change and degree of variation in skeletal integrity of live birds during the first cycle of egg laying. The humerus and tibia of White Leghorns were scanned repeatedly at 10-wk intervals from 15 to 65 wk of age. The BMD of the humerus increased from 15 to 65 wk of age, whereas the BMD and BMC of the tibia increased from 15 to 55 wk of age, resulting in a bone-by-age interaction (P < 0.001). The BMC of the humerus did not change from 15 to 55 wk of age but increased at 65 wk of age. Age-related increases in BMD and BMC may be due to the inability of dual energy X-ray absorptiometry to distinguish medullary from structural bone. The CV for BMD and BMC of egg-type chickens was greater than 10% after 25 wk of age, which suggested that bone densitometry could be used as an indicator tool in genetic selection with a potential for improving skeletal integrity of birds.

Assessing bone mineral density in vivo: dual energy X-ray absorptiometry.

Dual-energy X-ray absorptiometry can be used as a noninvasive tool to monitor the skeletal integrity of live birds. A pDexa X-ray bone densitometer was used to determine bone mineral densities (BMD) of the left tibia together with the fibula and the humerus of live, unanesthetized birds. Densitometry effectively detected changes in bone integrity of live birds fed varying levels of dietary calcium. Hens consuming 1.8, 3.6, or 5.4% dietary calcium had BMD of 0.147, 0.157, and 0.176 g/cm2 (SEM = 0.005), respectively (linear effect, P < 0.001). Likewise, bone ash weight, breaking force, stress, modulus of elasticity, and eggshell traits also increased linearly in response to increased calcium in the diet (P < 0.05). Densitometric live scans for BMD were positively correlated (P < 0.001) with bone breaking force (r = 0.65) and bone ash (r = 0.77). We also monitored BMD in live Leghorn and broiler females during their life cycle. The tibial BMD of White Leghorns and broilers increased from 15 to 65 wk of age with the BMD of the broiler tibia increasing at a greater rate than that of the Leghorn tibia (line x age interaction, P < 0.0001). A precipitous drop in BMD occurred during an induced molt of Leghorns subjected to 10 d of feed withdrawal. Our long-term goal is to improve skeletal integrity in egg-type chickens by genetic selection for improved BMD. By crossing a broiler with an egg-laying line, an F2 resource population of birds has been developed to identify quantitative trait loci influencing BMD in chickens.

Cross-species hybridisation of pig RNA to human nylon microarrays.

BACKGROUND: The objective of this research was to investigate the reproducibility of cross-species microarray hybridisation. Comparisons between same- and cross-species hybridisations were also made. Nine hybridisations between a single pig skeletal muscle RNA sample and three human cDNA nylon microarrays were completed. Three replicate hybridisations of two different amounts of pig RNA, and of human skeletal muscle RNA were completed on three additional microarrays. RESULTS: Reproducibility of microarray hybridisations of pig cDNA to human microarrays was high, as determined by Spearman and Pearson correlation coefficients and a Kappa statistic. Variability among replicate hybridisations was similar for human and pig data, indicating the reproducibility of results were not compromised in cross-species hybridisations. The concordance between data generated from hybridisations using pig and human skeletal muscle RNA was high, further supporting the use of human microarrays for the analysis of gene expression in the pig. No systematic effect of stripping and re-using nylon microarrays was found, and variability across microarrays was minimal. CONCLUSION: The majority of genes generated highly reproducible data in cross-species microarray hybridisations, although approximately 6% were identified as highly variable. Experimental designs that include at least three replicate hybridisations for each experimental treatment will enable the variability of individual genes to be considered appropriately. The use of cross-species microarray analysis looks promising. However, additional validation is needed to determine the specificity of cross-species hybridisations, and the validity of results.

A validated liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry method for quantitation of cocaine and benzoylecgonine in human plasma.

In order to support studies on various medication protocols for the treatment of cocaine abuse, an accurate, precise, and sensitive (2.5 to 750 ng/mL) liquid chromatography-tandem mass spectrometry assay was developed to determine cocaine and benzoylecgonine in human plasma. Cocaine-d3 and benzoylecgonine-d3 were added as internal standards and samples were subjected to solid-phase extraction. Cocaine recovery was 94.4% and benzoylecgonine was 80.3% at 2.5 ng/mL. The selected reaction monitoring of parent ions at m/z 304 and 290 resulted in strong fragments at m/z 182 and 168 for cocaine and benzoylecgonine, respectively. The method was fully validated. The mean measured concentration at the 2.5 ng/mL, the lower limit of quantitation, was within 10.8% of the target and the precision determined at the low (5 ng/mL), medium (50 ng/mL), and high (650 ng/mL) quality controls ranged from 0.9 to 6.2 %CV. Cocaine and benzoylecgonine concentrations in plasma treated with 1% NaF showed changes of less than 10% when maintained at room temperature for up to 7 h and no significant changes when subjected to three freeze-thaw cycles. The concentrations of cocaine and benzoylecgonine remained stable in plasma samples stored at -20 degrees C for up to 11 months. Methanolic stock solutions of both analytes are stable, staying within 2% of the freshly prepared stock solutions, when stored at -20 degrees C for up to 235 days. Both extracted analytes reconstituted in methanolic solutions are stable for up to seven days whether stored at -20 degrees C or at room temperature on the autosampler. The method is rugged, rapid, and robust and has been applied to the batch analysis of more than 700 samples during pharmacokinetic profiling to assess potential interactions between intravenous (i.v.) cocaine challenge and treament medications. Results from three of these subjects receiving 40 mg (i.v.) cocaine demonstrate the utility of the method.

Simultaneous determination of delta9-tetrahydrocannabinol and 11-nor-9-carboxy-delta9-tetrahydrocannabinol in human plasma by solid-phase extraction and gas chromatography-negative ion chemical ionization-mass spectrometry.

Delta9-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-delta9-tetrahydrocannabinol (THCA) in human plasma can be simultaneously detected using solid-phase extraction with gas chromatography and negative ion chemical ionization mass spectrometry. THC-d3 and THCA-d3 are added as internal standards; protein is precipitated with acetonitrile and the resulting supernatants diluted with 0.1 M sodium acetate (pH 7.0) prior to application to the solid-phase extraction columns. THC and THCA were eluted separately and then pooled, dried under air, and derivatized with trifluoroacetic anhydride and hexafluoroisopropanol. The derivatized THC-d0 gives abundant molecular anions (m/z 410), and the derivatized THCA-d0 gives abundant fragment ions (m/z 422) formed by loss of (CF3)2CHOH from its molecular anion. The recoveries of THC and THCA were 74% and 17%, respectively. The lower and upper limits of quantitation were 0.5 and 100 ng/mL for THC and 2.5 ng/mL and 100 ng/mL for THCA. The within-run accuracy and precision for THC (measured at 0.5, 1, 10 and 75 ng/mL) ranged from 98 to 106% (% target) and 4.1 to 9.5 (%CV), respectively. For THCA, the within-run accuracy and precision (measured at 2.5, 5, 10, and 75 ng/mL) ranged from 89 to 101% and 4.3 to 7.5%, respectively. The between-run accuracy and precision for THC ranged from 92 to 110% and 0.4 to 12.4%, respectively. The between-run accuracy and precision for THCA ranged from 97 to 103% and 6.5 to 12.3%, respectively. In processed samples stored in reconstituted form at -20 degrees C, THC and THCA were stable for at least three days. THC and THCA stored in plasma were stable following three freeze/thaw cycles. THC and THCA in whole blood at room temperature for 6 h, or in plasma stored at room temperature for 24 h, did not show significant change. Storage in polypropylene containers for 7 days at -20 degrees C and the presence of 1% sodium fluoride or the cannabinoid receptor antagonist, SR141716, at 1 microg/mL did not interfere with the quantitation of THC and THCA. In three individuals who smoked marijuana under controlled dosing conditions, peak THC concentrations of 151, 266, and 99 ng/mL were seen in the first plasma samples drawn immediately after the end of smoking, and corresponding peak THCA concentrations of 41, 52, and 17 ng/mL occurred at 0.33 to 1 h after cessation of smoking.

Differential N-demethylation of l-alpha-acetylmethadol (LAAM) and norLAAM by cytochrome P450s 2B6, 2C18, and 3A4.

Incubation of l-alpha-acetylmethadol (LAAM) or norLAAM with cDNA-expressed P450s 3A4, 2B6, and 2C18 produced significant N-demethylation products. P450s 2C19, 2C8, 3A5, 2C9, 3A7, 1A1, and 2D6 (norLAAM only), also produced detectable product. Coexpression of cytochrome b(5) enhanced LAAM N-demethylation, most dramatically for 3A4, but had marginal effects on norLAAM N-demethylation. Modeling total liver metabolism using immunoquantification and relative activity factors of P450s suggests contributions of P450 3A4 > 2B6 > 2C18, with the importance of 2B6 to 2C isozymes enhanced by relative activity factors. The ratio of dinorLAAM to norLAAM plus dinorLAAM formed from LAAM did not exceed 20%, and was isozyme and cytochrome b(5) coexpression dependent. This ratio decreased with concentration with 3A4, but was relatively constant for 2B6 and 2C18. The human liver microsomes substrate-concentration response was similar to cDNA-expressed 3A4, but the ratio was higher. Changes in the environment of cDNA-expressed 3A4 also effected the magnitude of the ratio, but not the concentration-dependent decrease. These studies show that the N-demethylation of LAAM and norLAAM is not restricted to P450 3A4, particularly P450s 2B6 and 2C18, and suggest that the mechanism of sequential metabolism for 3A4 differs from that of 2B6 and 2C18.

Evaluation of immunoassays for semiquantitative detection of cocaine and metabolites or heroin and metabolites in extracts of sweat patches.

Two types of immunoassays, radioimmunoassay (RIA) and microplate enzyme immunoassay (EIA), were compared for their ability to detect and quantitate cocaine and metabolites or heroin and metabolites in extracts of sweat patches. Experiments used sweat patches that had been fortified with cocaine, benzoylecgonine (BE), and ecgonine methyl ester (EME) or 6-acetylmorphine (6-AM), heroin, and morphine. Assays were first evaluated for sensitivity in detection of the analyte(s) known to be excreted in sweat (cocaine >> BE and EME; 6-AM > heroin > morphine). The cocaine metabolite RIA had cross-reactivity for cocaine > BE > EME, and the cocaine metabolite EIA had cross-reactivity for BE > cocaine >> EME. The RIA, having greater sensitivity for COC, was studied further. Optimal linearity was 4 to 200 ng/patch, and quantitation within these limits at 4, 75, and 150 ng/patch had intrarun %CVs within 7.8% and percent targets within 15% and inter-run %CVs within 13.5% and % targets within 13%. The opiate RIA had cross-reactivities for morphine >> 6-AM and heroin. The opiate EIA had cross-reactivities for 6-AM and heroin of 42 and 28% relative to morphine, respectively. The EIA, having greater sensitivity for 6-AM and heroin, was studied further. The limits of detection ranged from 1.7 to 24.7 ng/patch, and the lower limits of quantitation ranged from 7.3 ng/patch to beyond the linear range. The assay, however, had consistently good precision at 4 and 5 ng/patch, and optimal linearity was established from 4 to 100 ng/patch. With controls at 5, 25, and 90 ng/patch, both intrarun and inter-run precision were acceptable. Quantitation was accurate at 5 and 25 ng/patch, but the 90 ng/patch controls were consistently < 70% of target. Because our studies focused on the assays that had greater sensitivity for the analytes excreted in sweat, we did not fully evaluate the cocaine metabolite EIA or the RIA opiate screen and therefore cannot make any comment on the usefulness of these assays for detecting analytes in extracts of sweat patches beyond predicting that they will have less sensitivity. Both the cocaine metabolite RIA and opiate EIA had the ability to detect analytes known to be extracted from sweat patches.

Variables in human liver microsome preparation: impact on the kinetics of l-alpha-acetylmethadol (LAAM) n-demethylation and dextromethorphan O-demethylation.

We examined three primary variables in the preparation of human liver microsomes. In three experiments, each using three livers, we manipulated 1) the force of the first centrifugation (9,000, 10,500, or 12,000g); 2) the presence of sucrose in the homogenization buffer; and 3) the number of homogenizing strokes (6, 8, or 10). Sedimentation plots for the marker enzymes succinate dehydrogenase, NADPH cytochrome P450 reductase (reductase), and glutathione S-transferase in the resulting premicrosomal, microsomal, and cytosolic fractions suggest that enhanced purity of microsomes can be obtained by reducing force of centrifugation, including sucrose, and increasing the number of homogenization strokes. Each microsomal fraction was also assayed for protein content, cytochrome P450, NADH cytochrome b(5) reductase, cytochrome b(5), absorbance at 420, p-nitrophenol hydroxylation, tolbutamide hydroxylation, dextromethorphan N- and O-demethylation, glucuronidation of morphine and 1-naphthol, and ester cleavage of p-nitrophenolacetate. These microsomal indicators were ranked and tested for statistical differences. The use of 9000g statistically increased optimal recovery (per gram of liver) and specific activity (per milligram of protein). The inclusion of sucrose improved activity specific to reductase activity. Ten homogenization strokes improved activity specific to reductase activity. Substrate-dependent activities of dextromethorphan O-demethylation to dextrorphan and the N-demethylation of l-alpha-acetylmethadol (LAAM) to norLAAM and dinorLAAM were compared in microsomes prepared with or without sucrose and microsomes prepared using 9,000 or 12,000g force, respectively. No significant differences were found in the concentration-dependent activities. Variation of the methods used to prepare human liver microsomes can significantly affect the recovery and specific activity of microsomal components; however, they do not appear to affect enzyme kinetics.

Urinary excretion of 4-hydroxyamphetamine and amphetamine in male and female Sprague-Dawley and Dark Agouti rats following multiple doses of amphetamine.

Previous studies have demonstrated that the cytochrome P450 2D subfamily is involved in the 4-hydroxylation of amphetamine in the rat. These studies followed urinary levels of 4-hydroxyamphetamine and amphetamine after a single dose of amphetamine given with the P450 2D inhibitor quinidine or in P450 2D-deficient Dark Agouti (DA) rats. Multiple doses of amphetamine are used by humans and in experimental neurotoxicity studies of amphetamine. We hypothesized that the elimination of amphetamine (as opposed to 4-hydroxyamphetamine) will remain elevated in the DA rat after multiple doses, implying that no alternative pathway is induced to compensate for reduced 4-hydroxylation. Male and female Sprague-Dawley (SD) and DA rats were given daily i.p. injections of 5 mg/kg amphetamine for 5 days. Urine was collected at 12-h intervals and analyzed by HPLC for the presence of amphetamine and 4-hydroxyamphetamine. The amount of amphetamine detected in the urine remained elevated with a corresponding reduction of 4-hydroxyamphetamine in the DA rats when compared to SD rats over the entire time course. This reduction in 4-hydroxyamphetamine was greater in female than male DA rats; no difference was found between male and female SD rats. At the dose used, amphetamine did not increase with time and total amphetamine and 4-hydroxyamphetamine excreted by all rats was not different, implying no accumulation of amphetamine. These results suggest that no alternative pathway is induced following multiple doses of amphetamine in normal SD or P450 2D deficient DA rats.

Selective involvement of cytochrome P450 2D subfamily in in vivo 4-hydroxylation of amphetamine in rat.

The cytochrome P450 (P450) 2D subfamily catalyzes ring hydroxylation of amphetamines. We tested the hypothesis that P450 2D is selectively involved in amphetamine 4-hydroxylation. Urinary elimination of 4-hydroxyamphetamine and amphetamine was determined in male Sprague-Dawley rats pretreated with P450 inducers and inhibitors. The urinary 24-h metabolic ratio (amphetamine/4-hydroxyamphetamine) was not affected by the inducers 3-methylcholanthrene, isosafrole, phenobarbital, ethanol, pregnenolone-alpha-carbonitrile, and clofibrate. Isosafrole did, however, increase amphetamine elimination along with urine volume. Urinary elimination of 4-hydroxyamphetamine was significantly decreased by, and the metabolic ratio increased by, the inhibitors 1-aminobenzotriazole, CCl(4), quinidine, quinine, and primaquine. Diallyl sulfide and troleandomycin had no effect. In rat liver microsomes primaquine was shown to be an inhibitor of 2D activity. Urine 4-hydroxyamphetamine content correlated strongly (r(2) = 0. 989) with microsomal P450 2D activity in parallel-treated rats. These studies also substantiate that 4-hydroxylation of amphetamine is selectively performed by the P450 2D subfamily in the rat.

Detection of methadone, LAAM, and their metabolites by methadone immunoassays.

l-Alpha-acetylmethadol (LAAM) was recently approved as a substitute for methadone. LAAM, methadone, and their common metabolite, methadol, are extensively N-demethylated. The structural similarities of LAAM and its metabolites to methadone suggest that they may cross-react in methadone immunoassays. To test this hypothesis, drug-free urine was fortified with LAAM, norLAAM, dinorLAAM, methadol, normethadol, dinormethadol, methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), or 2-ethyl-5-methyl-3,3-diphenylpyrroline (EMDP) at 12 concentrations (0.03 to 100 microg/mL). Samples were analyzed using two enzyme immunoassays (Behring Diagnostics, EIA-b; Diagnostic Reagents, EIA-d); a fluorescent polarization immunoassay (Abbott, FPIA); two enzyme-linked immunosorbant immunoassays (Diagnostix, ELISA-d; STC Technologies, ELISA-s); a kinetic microparticles in solution immunoassay (Roche Diagnostic Systems, KIMS); and a radioimmunoassay (Diagnostic Products, RIA). LAAM had high cross-reactivity with ELISA-d (318.3%), RIA (249.5%), EIA-d (100.8%), KIMS (91.1%), and ELISA-s (75.3%). Methadol also displayed relatively high cross-reactivity as follows: EIA-d (97.8%), KIMS (85.4%), ELISA-d (70.3%), and FPIA (37.7%). Successive N-demethylations of LAAM and methadol were associated with loss of cross-reactivity. The methadone metabolites EDDP and EMDP showed little cross-reactivity. These findings suggest that LAAM use could result in positive immunoassay test results when using many of the commercially available methadone immunoassay kits and that confirmation of LAAM and its metabolites should be considered.

Enantioselective gas chromatography-negative ion chemical ionization mass spectrometry for methylphenidate in human plasma.

Therapeutic doses of Ritalin, a racemic mixture of d- and l-threo-methyphenidate, result in low plasma concentrations of methylphenidate. In order to assess the safety and efficacy of methylphenidate, a sensitive analytical method is needed. A gas chromatography-negative ion chemical ionization mass spectrometry (GC-NCI-MS) assay capable of measuring both d- and l-enantiomers in human plasma was developed and validated to support clinical studies involving administration of d,l-methylphenidate. d,l-Methylphenidate-d3 is added to 1-mL plasma samples. The plasma samples are made basic, mixed with isopropanol and extracted with hexane. The hexane extracts are then back-extracted into 0.1 N HCl. The acidified aqueous extract is made basic, cooled to ice temperature, and the methylphenidate derivatized with heptafluorobutyryl-l-prolyl chloride. The two diastereomeric derivatives are then extracted into hexane. The hexane extract is evaporated to dryness, reconstituted in ethyl acetate, and analyzed by GC-NCI-MS. This method can accurately (+/- 5% target) and precisely (< 11.1% coefficient of variation) quantitate enantiomers of threo-methylphenidate in human plasma and in the whole blood at concentrations ranging from 0.75 to 100 ng/mL. Plasma samples are stable for up to five freeze-thaw cycles when the duration of each cycle did not exceed 0.5 h. The drug degraded gradually when plasma samples were left at room temperature; a 6% loss at 3 h progressed to 17% at 12 h and to 35% at 24 h. Therefore, it is important that extraction of plasma samples begins within 0.5 h after samples are removed from the freezer. Whole blood stability results show that concentrations of methylphenidate in whole blood, with or without NaF, stored for up to 6 h at room temperature did not deviate from the target concentration by more than 13%. The derivatized methylphenidate in extract is stable at 4 degrees C for up to 10 days.

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.