Effects of temperature on evoked electrical activity and anoxic injury in CNS white matter.

Temperature is known to influence the extent of anoxic/ischemic injury in gray matter of the brain. We tested the hypothesis that small changes in temperature during anoxic exposure could affect the degree of functional injury seen in white matter, using the isolated rat optic nerve, a typical CNS white matter tract (Foster et al., 1982). Functional recovery after anoxia was monitored by quantitative assessment of the compound action potential (CAP) area. Small changes in ambient temperature, within a range of 32 to 42 degrees C, mildly affected the CAP of the optic nerve under normoxic conditions. Reducing the temperature to < 37 degrees C caused a reversible increase in the CAP area and in the latencies of all three CAP peaks; increasing the temperature to > 37 degrees C had opposite effects. Functional recovery of white matter following 60 min of anoxia was strongly influenced by temperature during the period of anoxia. The average recovery of the CAP, relative to control, after 60 min of anoxia administered at 37 degrees C was 35.4 +/- 7%; when the temperature was lowered by 2.5 degrees C (i.e., to 34.5 degrees C) for the period of anoxic exposure, the extent of functional recovery improved to 64.6 +/- 15% (p < 0.00001). Lowering the temperature to 32 degrees C during anoxic exposure for 60 min resulted in even greater functional recovery (100.5 +/- 14% of the control CAP area). Conversely, if temperature was increased to > 37 degrees C during anoxia, the functional outcome worsened, e.g., CAP recovery at 42 degrees C was 8.5 +/- 7% (p < 0.00001). Hypothermia (i.e., 32 degrees C) for 30 min immediately following anoxia at 37 degrees C did not improve the functional outcome. Many processes within the brain are temperature sensitive, including O2 consumption, and it is not clear which of these is most relevant to the observed effects of temperature on recovery of white matter from anoxic injury. Unlike the situation in gray matter, the temperature dependency of anoxic injury cannot be related to reduced release of excitotoxins like glutamate, because neurotransmitters play no role in the pathophysiology of anoxic damage in white matter (Ransom et al., 1990a). It is more likely that temperature affects the rate of ion transport by the Na(+)-Ca2+ exchanger, the transporter responsible for intracellular Ca2+ loading during anoxia in white matter, and/or the rate of some destructive intracellular enzymatic mechanism(s) activated by pathological increases in intracellular Ca2+.


Regional expression of c-fos and heat shock protein-70 mRNA following hypoxia-ischemia in immature rat brain.

Cerebral ischemia induces the expression of a number of proteins that may have an important influence on cellular injury. The purpose of this study was to compare the regional effects of hypoxia-ischemia on the expression of the proto-oncogene, c-fos, and the heat shock protein-70 (HSP-70) gene in developing brain. Unilateral hypoxia-ischemia was produced in the brain of immature rats (7, 15, and 23 days after birth) using a combination of carotid artery ligation and systemic hypoxia (8% O2). After recovery for 2 and 24 h, the regional expression of c-fos and HSP-70 mRNA was determined using in situ hybridization. Littermates were permitted to recover for 1 week for assessment of histologic injury. Hypoxia-ischemia increased the expression of both c-fos and HSP-70 mRNA, but the topography of expression varied with the age of the animal as well as the mRNA species. In the 7-day-old group, expression of c-fos at 2 h increased in multiple regions of the ipsilateral hemisphere in nearly one-half of the animals, while HSP-70 mRNA was not expressed until 24 h and, then, predominantly in the hippocampus. In 15- and 23-day-old rats, expression of c-fos was increased at 2 h in the entorhinal cortex and in the dendritic field of the upper blade of the hippocampal dentate gyrus, while HSP-70 mRNA was prominently expressed in neocortex and the cell layers of the hippocampus. Interestingly, the strong expression of HSP-70 mRNA in dentate granule cells did not occur in the innermost layer of cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Urinary incontinence in older adults.

A frequent and largely neglected problem, incontinence in the elderly can often be ameliorated or cured. Transient incontinence is usually secondary to intercurrent disease and thus should be addressed by treatment of that pathology. Established incontinence is most often due to detrusor hyperactivity. Behavioral intervention and bladder relaxants are management options.


Estimation of aqueous solubility of organic molecules by the group contribution approach. Application to the study of biodegradation.

A reliable and generally applicable aqueous solubility estimation method for organic compounds based on a group contribution approach has been developed. Two models have been established based on two different sets of parameters. One has a higher accuracy, while the other has a more general applicability. The prediction potentials of these two models have been evaluated through cross-validation experiments. For model I, the mean cross-validated r2 and SD for 10 such cross-validation experiments were 0.946 and 0.503 log units, respectively. While for model II, they were 0.953 and 0.546 log units, respectively. Applying our models to estimate the water solubility values for the compounds in an independent test set, we found that model I can be applied to 13 out of 21 compounds with a SD equal to 0.58 log unit and model II can be applied to all the 21 compounds with a SD equal to 1.25 log units. Our models compare favorably to all the current available water estimation methods. A program based on this approach has been written in FORTRAN77 and is currently running on a VAX/VMS system. The program can be applied to estimate the water solubility of the water solubility of any organic chemical with a good or fairly good accuracy except for except for electrolytes. Applying our aqueous solubility estimation models to biodegradation studies, we found that although the water solubility was not the sole factor controlling the rate of biodegradation, ring compounds with greater solubilities were more likely to biodegrade at a faster rate. The significance of the relationship between water solubility and biodegradation activity has been illustrated by predicting the biodegradation activity of 27 new chemicals based solely on their estimated solubility values.


Left ventricular hypertrophy and diastolic dysfunction.

The hypertrophied heart generally preserves systolic function, but it pays a price in diastolic dysfunction. Coronary reserve is impaired and susceptibility to myocardial ischemia increased. The pathophysiology is managed by addressing causes of pressure overload, preventing tachycardia, or, of course, by specific strategies to reduce or eliminate ischemia.


The Concise Connection Table: collected definitions with extensions for stereochemistry and saccharides.

The Concise Connection Table is a representation of molecular structures based on an implicit hierarchy of substructural components. This paper introduces a number of enhancements and extensions to the original definition, particularly relating to representations of stereochemistry and saccharides, and summarizes the overall principles of the CCT in one definitive presentation.


Do vascular wall cytokines promote atherogenesis?

Protein mediators commonly associated with inflammatory and immune responses may also convey signals among vascular endothelial and smooth muscle cells, thereby mediating the evolution of hyperplastic arterial disease. A picture is emerging in which cytokine expression, lipoprotein modification, and atheroma development are interlocking processes.


Agreement between parents' reports and adolescents' self-reports of problem behavior.

Parents' reports and adolescents' self-reports of problem behaviors in 883 11-19-year-olds from the general population were compared. Correlations between both informants' CBCL syndrome scores ranged from 0.27 to 0.56. Adolescents reported many more problems than their parents did about them. Discrepancies were larger for externalizing than for internalizing problems, were larger for girls than for boys and increased with age. The findings indicated that adolescents, especially as they grow older, are indispensable informants on their own problem behaviors.


Obsessive-compulsive disorder in childhood and adolescence: a clinical study.

The clinical records of 72 children and adolescents aged 5-18 with a diagnosis of OCD were examined. Mean age of onset was 11 years. Repeating, cleaning and checking were the most common compulsions. Twenty percent of subjects showed obsessions unrelated to compulsions. In 53% of cases stress situations preceded the disorder. Seventy-seven percent of subjects suffered some other psychiatric disorder, lifetime or current, particularly anxiety and affective disorders. The majority (57%) had some first-degree relative with a psychiatric diagnosis. Family conflicts, social withdrawal and poor school performance were also common features.


Attainments of severely mentally retarded adolescents by aetiology.

Piagetian cognitive level, language abilities, adaptive behaviour and graphic skills were assessed in three groups of adolescents with severe learning difficulties: Down's syndrome (DS), congenital cerebral palsy (CP) and unknown origin (NSP). Results were compared with assessments made seven years earlier. A wide range of scores was observed in each group. Medians of the DS and NSP groups were similar and generally were significantly higher than those of the CP group. When the number and severity of other impairments and disabilities (HDCP) was taken into account, group averages of attainments and progress were very similar. Implications for intervention are discussed.


Family change, parental discord and early offending.

The relationship between exposure to family change, exposure to parental discord during the period from birth to 10 years and risks of offending by the age of 13 years was studied in a birth cohort of New Zealand children. This analysis showed that while exposure to parental discord during middle and early childhood led to increased risks of early offending, exposure to family change in the absence of parental discord did not lead to increased risks of offending. The results also suggested that children with a history of early conduct problems were particularly susceptible to parental discord but that the effects of discord did not vary with the child's gender. These results persisted when errors of measurement in the reporting of offending were taken into account using latent class methods.


Pre- and perinatal factors and the risk of subsequent referral for hyperactivity.

Possible pre- and perinatal risk factors for subsequent referral for hyperactivity were assessed by comparing birth records of 129 referrals with the remaining 24,656 members of a geographically defined birth cohort. Relationships between the risk factors were accounted for using logistic regression methods. The significant factors were: social class, maternal age, antepartum haemorrhage, length of labour (second stage), 1-min Apgar and sex. Associations between referral for hyperactivity and the pregnancy, labour and birth outcome factors were not explained by the socio-demographic variables. The results suggest that such factors have a statistically significant association with referral for hyperactivity and may be of modest aetiological importance. However, the predictive power of the final set of factors remained low even on the original data set.


The effect of delay on hyperactive and non-hyperactive children's response times: a research note.

Twenty-four hyperactive and 55 non-hyperactive children made a button press after the disappearance of a stimulus presented for either 1, 15 or 30 sec. Hyperactive children's responses were generally slower than those of non-hyperactive children and increased with length of pre-response delay, while non-hyperactive children's response time remained the same across all presentation levels. The results are interpreted as giving support to accounts that stress the role of pre-response delay, rather than time on task per se, as an important determinant of hyperactive children's attentional performance.


Children's drawings of topics differing in emotional significance--effects on placement relative to a self-drawing: a research note.

Children aged 4-6 years were asked to add drawings of someone they liked and someone they disliked to a picture of themselves. Liked figures were drawn closer than disliked figures to the self-figure only when the procedure was standardized and controlled so as to equate the tasks of positioning each of the added figures. With unmatched positioning tasks there was no effect of significance of figure on placement. The data demonstrate that a child's emotional attitude to a figure can affect its placement in a drawing, but that the effect is weak and easily masked.


Understanding drawings and beliefs: a further test of the metarepresentation theory of autism: a research note.

Leslie (1987, Psychological Review, 94, 412-426) proposed that the "theory of mind" deficit in autism was the result of a metarepresentation impairment. Studies employing False Photograph or Belief tests have shown that in autism the deficit is restricted to representing mental representations, and does not extend to representing pictorial representations. In this study, we tested this claim further using a False Drawing test. Subjects with autism performed at the same level as mentally handicapped or normal 4-year-old subjects on the False Drawing test, but significantly worse on the False Belief test. This confirmed the specificity of the deficit in autism.


Fears and fearfulness in children and adolescents: a genetic analysis of twin data.

A sample of 175 same sex dizygotic pairs and 144 monozygotic twin pairs aged between 8:00 and 18:00 completed the Fear Survey Schedule for Children--Revised. The heritabilities were significant for Fear of the Unknown (h2 = 0.46, p less than 0.001), Fear of Injury and Small Animals (h2 = 0.46, p less than 0.001), Fear of Danger (h2 = 0.34, p less than 0.001) and for Total Fear Score (h2 = 0.29, p less than 0.001). Multiple regression was used to estimate the heritability of extreme fearfulness (h2g). For each of the fear factors the values of h2g were of similar magnitude to those of h2 suggesting that there is no evidence of greater genetic influences at more extreme levels of fearfulness.


Psychometric evaluation of a revised fear survey schedule for children and adolescents.

This study describes the second revision of a fear survey schedule for children which was originally developed by Scherer and Nakamura in the 1960's. The revised instrument (FSSC-II) was psychometrically evaluated on a sample of 918 children and adolescents aged between 7 and 18 years, attending regular primary and secondary schools in urban, suburban and rural areas of Victoria. It was demonstrated to have high internal consistency. The convergent and divergent validity of the revised instrument was examined by correlating it with conceptually related as well as distinct scales, respectively. Such analyses demonstrated sound validity. A five-factor solution almost identical to that reported for the FSSC-R, is described as are age and gender differences. The most common fears on the revised instrument are also reported.


Sources of distress among New Zealand adolescents.

This study examined sources of distress experienced by 15-year-old adolescents in a large sample from the general population. We identified four types of stressful life circumstances relating to problems of self-image and independence, academic and physical competence, parental conflict, and moving residence and schools. Girls reported higher levels of distress for the first three types of circumstance. Reports of distress were associated with poor family social support, maternal depression and parental separation. Both DSM-III disorder and poor social competence were associated with differential patterns of distress. Lastly, poor social competence and high distress were independent and additive predictors of mental health disorders.


Out of sight or out of mind? Another look at deception in autism.

The penny-hiding game is a deception game that occurs naturally in parent-child and child-child interaction. It involves minimal linguistic demands, and is lots of fun. Oswald and Ollendick (1989) employed it with subjects with autism and reported an impaired capacity for deception. They also found that this correlated with performance on both a false belief ("theory of mind") test as well as various measures of social behaviour. The experiment reported here set out to replicate Oswald and Ollendick's important results, and then extend them by using a new technique for error analysis. We succeeded in replicating the autism-specific deception impairment as well as the finding that deception capacity correlates highly with performance on a false belief test. In addition, the new analytic technique discriminated the group with autism from controls more clearly than the traditional index of deception. Specifically, subjects with autism, whilst fully capable of enjoying the game as a game of object occlusion (keeping things out of sight), failed to perceive the game as a game of information occlusion (keeping things out of mind), unlike normal children or subjects with a mental handicap of an equivalent or lower mental age. The dissociation in autism between occluding objects vs occluding information is discussed in relation to other research showing that subjects with autism are impaired in understanding the principle that "seeing leads to knowing".


Comprehension of affect in context in children with pervasive developmental disorders.

Fifteen children with Pervasive Developmental Disorders (PDD) (mean age 12.7 years) were compared to mental age matched normal children on matching a context to its appropriate emotion. PDD children were slightly but significantly impaired on this task relative to a non-social task equated for difficulty. Both matching tasks were highly correlated with cognitive variables; the social matching task alone was correlated with social skill level, and neither task was correlated with ratings of social deviance. Results are discussed in terms of the demands of social cognitive tasks, the magnitude of social cognitive findings, control group selection and individual differences.


Understanding of simple and complex emotions in non-retarded children with autism.

Non-retarded autistic children are compared to normal controls on measures of emotion expression and recognition. In general, autistic subjects recounted appropriate examples of simple and complex emotions, and accurately labeled relatively ambiguous affect expression in pictures. Autistic children manifested some difficulty talking about socially derived emotions, pride and embarrassment. They required more time and more prompts, their responses were more tentative and "scripted", and they displayed limited understanding of the salience of others in embarrassing situations. Results are discussed in relation to theory of mind impairment and compensation strategies in autism.


Children's understanding of the feeling experience and causes of sympathy.

German children in five age groups, 5-6, 7-8, 9-10, 11-12 and 14-15 years, were interviewed about feeling experiences of sympathy and situations that elicit sympathy. While the younger children focussed on the emotion of sadness, the older children increasingly described sympathy as a multi-dimensional emotional experience consisting of sadness, desire to help, and preoccupied thoughts about the other in distress. The most marked developmental change occurred in the frequency of reference to preoccupied thoughts. The older children regarded severe life-threatening distress as situations that elicit sympathy, whereas the younger children spontaneously named everyday life distress or favourite animals.


Early channels of mother-infant communication: preterm and term infants.

The impact of prematurity on the responsiveness of mothers and their 4-month-old infants was examined across three channels of communication: attentional, vocal and affective. Log-linear models were used to determine how the behavior of one partner was conditional upon the behavior of the other during home observations of 24 preterm and 24 term infants and their mothers. Visual attention was elicited by vocalization, and the onset of infant gaze was marked by a maternal smile. Mothers and infants responded to vocalizations with vocalizations, and mothers responded to smiles with smiles. Mothers of preterm infants were particularly responsive to their infants' signals within the attentional, vocal and affective channels. Preterm infants demonstrated correspondingly heightened responsiveness within the vocal and affective channels.


Soft object and pacifier attachments in young children: the role of security of attachment to the mother.

A longitudinal study was conducted to evaluate the hypothesis that a healthy mother-infant relationship is a prerequisite for the development of a child's attachment to a blanket or other soft object. Security of attachment to mother was measured at 12 months with the Strange Situation Test and at 30 months with the Attachment Q-Sort. Most of the children with soft object attachments were rated as securely attached to their mothers at both ages. Children with pacifier attachments, on the other hand, were less often rated as securely attached and were more likely to show changes in security classification between 12 and 30 months. The results are discussed in terms of necessary, but not sufficient, conditions for the development of attachments to inanimate objects.


Methylphenidate and cognitive perseveration in hyperactive children.

Effects of methylphenidate on cognitive flexibility were investigated in 26 children with Attention Deficit Hyperactivity Disorder (ADHD), by assessing perseverative errors on the Wisconsin Card Sorting Test (WCST) and the emergence of clinical symptoms indicative of cognitive perseveration. A double-blind, placebo-control, randomized crossover design was used in which each child was assessed twice in each drug condition (placebo, 0.3 and 1.0 mg/kg). Results indicated that methylphenidate increased perseverative errors on the first assessment but decreased them on the second; clinical symptoms of perseveration occurred at both assessments. Findings suggest that MPH may reduce cognitive flexibility temporarily in some ADHD children.


Gender ratios among reading-disabled children and their siblings as a function of parental impairment.

Gender ratios are reported for 374 reading-disabled probands and their 530 siblings included in five independent studies of reading disability. Ratios were tabulated for each study as a function of parental impairment (neither parent affected, mother only affected, father only affected, and both parents affected). Results reveal a small excess of male probands in referred and clinic samples of reading-disabled children, but not in research-identified samples. Gender ratios among siblings of reading-disabled probands are approximately 1:1. In addition, combined results indicate that gender ratios of neither probands nor their siblings vary substantially as a function of parental impairment.


Rising IQ scores in British children: recent evidence.

Between 1987 and 1990 IQ scores for 333 5-year-old white school children were obtained using the Wechsler Pre-School and Primary Scale of Intelligence (WPPSI) and the results were compared with 112 children assessed in 1967. The mean full scale IQ score of 113.3 was 8 points higher than the mean of 105.1 in the 1967 study. These findings indicate that the rate of increase of IQ scores was comparable and probably higher than that reported in the U.S.A. for the period 1932-78. It is important that this large increase is taken into account for correct interpretation of IQ test results.


Toddlers' sleep and temperament: reporting bias or a valid link? A research note.

Objective sleep measures derived from a computerized movement detector attached to the child's leg, were obtained for a group of 31 toddlers (mean age: 18.5 months). The monitored sleep parameters were compared with maternal assessment of the child's sleep and her perception of his temperament. The validity of maternal diaries as a measure of the child's sleep was not supported. Nevertheless, a link between both subjective and objective sleep measures with temperament dimensions was indicated. The modest association between sleep and temperament may suggest either a continuity between some aspects of day- and night-time functioning or, alternatively, the influence of the child's sleep behavior in shaping the mother's perceptions of her toddler's temperament.


Parent-teacher agreement on kindergarteners' behavior problems: a research note.

The present study investigated parameters of agreement as well of disagreement among mothers' and teachers' ratings of the behavior of kindergarten boys and girls. Findings indicated a generally low to moderate level of agreement between these two types of informants. They also suggested that demographic and background characteristics can effect size and direction of level of agreement. Although proportions of children rated as having the listed behavior problems varied in magnitude according to mothers and teachers, the rank order of the proportion of children with those behavior problems was rather similar for both informants. Such similar rankings of prevalence rates may reflect parents' and teachers' agreement on the consistency of children's behavior across the home and school settings.


Drug level monitoring: antidepressants.

The determination of antidepressant drugs which act by blocking neuronal uptake of biogenic amines, because of their widespread use and high toxicity, remains one of the most commonly requested drug assays in clinical laboratories. Easy to use immunoassay reagents for the estimation of these drugs are commercially available. However, immunoassays have not been universally accepted because of high probability of these reagents producing false negative and false positive results. At present, column liquid chromatography with absorbance detection and coupled with solid-phase extraction is the most viable technique for a general procedure for the identification and determination of these drugs. The technique of liquid chromatography is economical, environmental friendly since water-miscible and biodegradable solvents can be used for extraction of drugs and their chromatographic separation, and amenable to full automation. New techniques of separation, such as supercritical fluid chromatography and capillary zone electrophoresis, have not yet been applied for the determination of therapeutic concentrations of antidepressants.


Simultaneous determination of stable isotopically labelled L-histidine and urocanic acid in human plasma by stable isotope dilution mass spectrometry.

A capillary gas chromatographic-mass spectrometric method for the simultaneous determination of stable isotopically labelled L-histidine (L-[3,3-2H2,1',3'-15N2]histidine, L-His-[M + 4]) and urocanic acid ([3-2H,1',3'-15N2]urocanic acid, UA-[M + 3]) in human plasma was developed using DL-[2,3,3,5'-2H4,2'-13C,1',3'-15N2]histidine (DL-His-[M + 7]) and [2,3,5'-2H3,2'-13C,1',3'-15N2]urocanic acid (UA-[M + 6]) as internal standards. L-Histidine and urocanic acid were derivatized to alpha N-(trifluoroacetyl)-imN-(ethoxycarbonyl)-L-histidine n-butyl ester and imN-(ethoxycarbonyl)urocanic acid n-butyl ester. Quantification was carried out by selected ion monitoring of the molecular ions of the respective derivatives of L-His-[M + 4], DL-His-[M + 7], UA-[M + 3] and UA-[M + 6]. The sensitivity, specificity, precision and accuracy of the method were demonstrated to be satisfactory for measuring plasma concentrations of L-His-[M + 4] and UA-[M + 3] following administration of trace amounts of L-His-[M + 4] to humans.


Excretion of formaldehyde, malondialdehyde, acetaldehyde and acetone in the urine of rats in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin, paraquat, endrin and carbon tetrachloride.

Formaldehyde (FA), acetaldehyde (ACT), malondialdehyde (MDA) and acetone (ACON) were simultaneously identified in urine, and their excretion quantitated in response to chemically induced oxidative stress. Urine samples of female Sprague-Dawley rats were collected over dry ice and derivatized with 2,4-dinitrophenylhydrazine. The hydrazones of the four lipid metabolic products were quantitated by high-performance liquid chromatography on a Waters 10-microns mu-Bondapak C18 column. The identities of FA, ACT, MDA and ACON in urine were confirmed by gas chromatography-mass spectrometry. An oxidative stress was induced by orally administering 100 micrograms/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin, 75 mg/kg paraquat, 6 mg/kg endrin or 2.5 ml/kg carbon tetrachloride to rats. Urinary excretion of FA, ACT, MDA and ACON increased relative to control animals 24 h after treatment with all xenobiotics. The system has wide-spread applicability to the investigation of altered lipid metabolism in disease states and exposure to environmental pollutants.


Identification of 4-hydroxyandrost-4-ene-3,17-dione metabolites in prostatic cancer patients by liquid chromatography-mass spectrometry.

Liquid chromatography with thermospray mass spectrometry has proved to be an invaluable technique for the study of metabolic degradation of xenobiotics in complex biological fluids. This paper describes the detection of 4-hydroxyandrost-4-ene-3,17-dione and its metabolites in urinary extracts from prostatic cancer patients. Several metabolites were detected including 4 beta,5 alpha-dihydroxyandrostan-3,17-dione, 3,17-dihydroxyandrostan-4-ones and 3 alpha-hydroxy-5 beta-androstan-4,17-dione.


Preparation of an optimum mobile phase for the simultaneous determination of neurochemicals in mouse brain tissues by high-performance liquid chromatography with electrochemical detection.

A systematic method is described for the optimization of a mobile phase for the simultaneous determination of 24 neurochemicals consisting of catecholamine, serotonin, their precursors and metabolites and related materials. This mobile phase contained sodium acetate (0.04 M), citric acid (0.01 M), sodium chloride (0.0126 M), sodium octyl sulfate (91 mg/l), tetrasodium EDTA (50 mg/l) and 10% (v/v) methanol. When this optimum mobile phase was applied to the analysis of brain tissues of the Swiss male mouse, twelve neurochemicals were quantified in the free state: tyrosine, L-beta-3,4-dihydroxyphenylanine, dopamine, 3,4-dihydroxyphenylacetic acid, 4-hydroxy-3-methoxyphenylacetic acid, norepinephrine, 3-methoxy-4-hydroxyphenylglycol, DL-3,4-dihydroxymandelic acid, DL-4-hydroxy-3-methoxymandelic acid, serotonin, L-tryptophan, 5-hydroxyindole-3-acetic acid and DL-synephrine and normetanephrine, appearing as a fused peak. This fused peak was present on the chromatogram tracings of all the mouse brain tissues. The separable neurochemicals not found by this procedure in the Swiss male mouse tissues were DL-3,4-dihydroxyphenylglycol,5-hydroxytryptophan, epinephrine, DL-octopamine, metanephrine, deoxyepinephrine, homovanillyl alcohol, N-acetylserotonin, tyramine and 3-methyltyramine.


Electrochemical determination of femtomole amounts of free reduced and oxidized glutathione. Application to human hair follicles.

A simple and sensitive high-performance liquid chromatographic technique has been developed for the quantification of free reduced and free oxidized glutathione in biological samples. After acidic extraction and isocratic separation of the compounds of interest on a reversed-phase column, both forms of glutathione are quantified with a coulometric detector working in the oxidative mode. The limit of detection is 125 fmol for reduced glutathione and 400 fmol for the oxidized form (signal-to-noise ratio of 3). This sensitivity allows the measurement of the small amount of glutathione present in a single hair follicle. The technique is well adapted to microsamples, i.e. for non-invasive sampling technique (hair, skin, tears, etc.) and can be adapted to various cells or tissues.


High-performance liquid chromatographic determination of selenocysteine with the fluorescent reagent, N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid.

The method described is based on derivatization of selenocysteine with N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid and responds linearly to selenocysteine spiked into plasma. Recovery is insensitive to inter-individual variation or use of serum versus plasma, but is decreased by hemolysis. The derivative is stable for at least three days. The total imprecision of determinations in plasma was 0.8-2.1% (coefficient of variation) over the range of 6-30 microM selenocysteine, with a detection limit of 0.4 microM (3 x S.D.). There was no significant interference from plasma thiols. This appears to be the first report of the selective reaction of free selenocysteine with a fluorescent reagent. This simple method works well in plasma and serum and may be adaptable to other types of samples.


Resolution of serum aluminum-binding proteins by size-exclusion chromatography: identification of a new carrier of aluminum in human serum.

We have examined the use of TSK-GEL HW 55S for determining the distribution of aluminum in human serum by size-exclusion chromatography (SEC). In comparison to other SEC matrices, this material has less affinity for ionized aluminum and separates serum proteins and their aluminum complexes with greater resolution. This enabled the identification of a previously unknown protein carrier, provisionally called albindin, that binds aluminum with great stability. Albindin appears to be distinct from the previously described aluminum carriers albumin and transferrin and may be important in the pathogenesis of disease secondary to hyperaluminemia.


Separation of rat pancreatic secretory proteins by cation-exchange fast protein liquid chromatography.

Rat pancreatic secretory proteins were separated by an automated liquid chromatography system utilizing a Mono S cation-exchange column. Optimal resolution was obtained with a multistep salt and pH gradient (0.01-2 M LiCl, pH 5.3-63). A total of fourteen well-separated peaks, as well as several minor peaks, were detected by UV absorption. The main pancreatic enzymes were resolved (two amylases, two chymotrypsinogens, two trypsinogens, proelastase, lipase, prophospholipase A2, procarboxypeptidase A, procarboxypeptidase B, and ribonuclease). In addition, proteins without enzymic activity, such as lithostathine and pancreatitis-associated protein, were identified. Activation of proenzymes did not occur during the separation. At a flow-rate of 0.5 ml/min, ca. 250 micrograms to 5 mg of protein could be applied with equal resolution. The reproducibility of retention volumes and peak areas was high (less than 1% or 5% variation, respectively). When radiolabeled proteins were separated, a comparable pattern of peaks was obtained. The technique described is, therefore, not only useful for analytical and preparative separation of pancreatic proteins but can additionally serve for quantitative determination of the pancreatic isoenzyme pattern.


Determination of cobalt in urine by gas chromatography-mass spectrometry employing nickel as an internal standard.

A gas chromatographic-mass spectrometric method for the determination of cobalt in biological materials employing stable enriched 62Ni as an internal standard and using lithium bis(trifluoroethyl)dithiocarbamate as a chelating agent is described. The method involves the addition of a known amount (1 microgram) of 62Ni to the sample, the formation of the chelate and the determination by selected-ion monitoring of the m/z 571/574 ratio, which corresponds to 59Co/62Ni. No appreciable memory effect was observed, and an acceptable dynamic range of 100 was found. There was good agreement between the cobalt concentration values determined by gas chromatography-mass spectrometry and electrothermal atomic absorption spectrometry. The present method has high sensitivity and can be used for the quantitation of cobalt at concentrations as low as 1 microgram/l. The use of enriched 62Ni circumvents the problem caused by endogenous nickel and simultaneously provides data on the nickel concentration in the biological sample without any additional experimental effort.


Determination of ceftazidime in dolphin serum by liquid chromatography with ultraviolet-visible detection and confirmation by thermospray liquid chromatography-mass spectrometry.

A simple and sensitive liquid chromatographic method has been developed for the determination of therapeutic levels of ceftazidime in dolphin serum. The method involved an ultrafiltration of diluted serum with an equal amount of acetonitrile-ethanol-water (40:40:20, v/v/v) through a 10,000 daltons molecular mass cut-off filter. Separation of ceftazidime from the other serum components was performed by ion-paired (dodecanesulfonate) liquid chromatography using a reversed-phase column eluted with acetonitrile-water solution. The ultraviolet absorbance of the column effluent was monitored in the 200-340 nm range of a photodiode-array detector or at 258.8 nm on a variable-wavelength ultraviolet-visible detector. Recoveries of ceftazidime from dolphin serum spiked with 20 and 2 micrograms/ml were 92.9 and 91.1% with coefficients of variation of 5.5 and 5.7%, respectively. A correlation coefficient of 0.9994 occurred with ceftazidime in aqueous solutions (n = 6, in duplicates). The limit of detection for this antibiotic was estimated to be approximately 50 ppb (ng/ml). The unbound ceftazidime concentrations in dosed dolphin serum were determined to calculate the protein bindings of this antibiotic which yielded 32 +/- 2%. The ceftazidime peak identity in dosed dolphin serum was confirmed by thermospray liquid chromatography-mass spectrometry. The thermospray mass spectrum of ceftazidime exhibited only the fragment ions, involving the opening of the beta-lactam ring, at m/z 237, 255 and 315 when positive-ion detection mode was employed and the fragment ions at m/z 235, 253 and 313 when negative-ion detection mode was used.


Determination of penicillin G in bovine plasma by high-performance liquid chromatography after pre-column derivatization.

A simple, selective, and sensitive liquid chromatographic method with ultraviolet detection was developed for the analysis of penicillin G in bovine plasma. The assay utilizes a simple extraction of penicillin G from plasma (with a known amount of penicillin V added as internal standard) with water, dilute sulphuric acid and sodium tungstate solutions, followed by concentration on a conditioned C18 solid-phase extraction column. After elution with 500 microliters of elution solution, the penicillins are derivatized with 500 microliters of 1,2,4-triazole-mercuric chloride solution at 65 degrees C for 30 min. The penicillin-mercury mercaptide complexes are separated by reversed-phase liquid chromatography on a C18 column. The method, which has a detection limit of 5 ng/ml (ppb) in bovine plasma, was used to quantitatively measure the concentrations of penicillin G in plasma of steers at a series of intervals after the intramuscular administration of a commercial formulation of procaine penicillin G.


High-performance liquid chromatographic determination of sotalol in plasma. I. Application to the disposition of sotalol enantiomers in humans.

Two high-performance liquid chromatographic analytical methods have been developed for the measurement of dl-sotalol or d-sotalol and l-sotalol in plasma, using dl-atenolol as internal standard. Quantitation of dl-sotalol was carried out, following solid-phase extraction, on a 5-microns C18 reversed-phase column, with a mobile phase containing acetonitrile, ion-pairing reagent and distilled water, using ultraviolet detection at 235 nm. Quantitation of d-sotalol and l-sotalol was based on derivatisation with the chiral agent S-(-)-alpha-methylbenzyl isocyanate, followed by chromatographic separation on a 3-microns C18 reversed-phase column, with a mobile phase containing methanol, glacial acetic acid and distilled water, with fluorimetric detection at 220 nm excitation and 300 nm emission. A preliminary application of the latter method suggests that the disposition of sotalol in humans is not enantioselective.


Intra-injector methylation of free fatty acids from aerobically and anaerobically cultured Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus.

Free fatty acids from the type strains of anaerobically and aerobically broth-cultured Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus cells were Soxhlet-extracted with hexane. The fatty acids were identified and quantified by gas chromatography and gas chromatography-mass spectrometry after intra-injector derivation with trimethylanilinium hydroxide. This derivatization method, which we propose as suitable for routine use in clinical microbiology, is fast, accurate and sensitive, with low toxicity. Whereas the fatty acid content of A. actinomycetemcomitans was affected by the cultivation atmosphere, i.e. C16:1, decreased under aerobic growth and C16:0 increased, that of the closely related H. aphrophilus was more stable.


Difference in the concentration of tryptophan metabolites between maternal and umbilical foetal blood.

Maternal and umbilical foetal blood at delivery were analysed for tryptophan metabolites by using fully automated high-performance liquid chromatography. The metabolites detected in 100 microliters of maternal plasma were kynurenine, serotonin, 5-hydroxyindoleacetic acid, indolelactic acid, indoleacetic acid and indolepropionic acid. These metabolites were present in various amounts in the protein-bound form. Except for indolepropionic acid, the concentrations of tryptophan metabolites were significantly higher in umbilical foetal plasma than in maternal plasma. In addition, 3-hydroxyanthranilic acid was present in umbilical foetal blood, but not in maternal blood. Furthermore, kynurenic acid was also detected in amniotic fluids.


Resolution of three forms of superoxide dismutase by immobilised metal affinity chromatography.

A new method for separation of three forms of superoxide dismutase (SOD) using immobilised metal affinity chromatography (IMAC) is reported. Fe-, Mn- and Cu/Zn-SODs were eluted sequentially from Cu(2+)-IMAC column with an increasing gradient of a counter ion (NH+4) run in combination with an increasing pH gradient (6.8-7.8). The combined gradient elution method resulted in separation of SODs with high resolution, the three proteins being eluted in electrophoretically homogeneous forms. Similar preparation could not be achieved by either increasing gradient of a counter ion or decreasing pH gradients used separately. The described methodology has been successfully applied for separation of three SODs from a protozoan parasite, indicating that this combined gradient elution system for IMAC offers new possibilities for the high-resolution separation of proteins exhibiting only minor differences in their amino acid composition and structure.


Separation of globins using free zone capillary electrophoresis.

This paper describes the use of high-performance capillary electrophoresis for the separation of globin chains. Adult and newborn haemolysates from normal individuals and children suspected of having thalassaemia were analysed using free zone electrophoresis. Separation of globins was accomplished using a 25 mM phosphate buffer at pH 11.8. Distinct peaks of alpha-, beta- and gamma-chains were resolved within 8 min. The coefficient of variation for within-day and between-day runs was 4.1% and 5.7%, respectively. This method is simple and rapid, and it can be used to screen for thalassaemia and for the clinical study of various haemoglobinopathies.


High-performance liquid chromatographic determination of navelbine in MO4 mouse fibrosarcoma cells and biological fluids.

A high-performance liquid chromatographic method is described for separating and determining navelbine and possible metabolites in plasma, cell culture medium and MO4 cells. Navelbine is extracted from these fluids by ion-pair extraction with sodium octylsulphate as the counter-ion at pH 3. The system uses a cyano column as the stationary phase and a mobile phase of acetonitrile-0.12 M phosphate buffer (pH 3) (60:40, v/v). Application of the method to a study of the pharmacokinetic behaviour of navelbine in MO4 mouse fibrosarcoma cells is reported.


Quantitation of a new cholecystokinin and gastrin receptor antagonist (L-365,260) in dog and rat plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) procedure has been developed for the quantification of L-365,260 (I), a cholecystokinin and gastrin receptor antagonist, in dog and rat plasma. The method involves liquid-liquid extraction and HPLC with ultraviolet detection. Standard curves were linear over the range 7.5-2000 ng/ml for rat and dog plasma. The method is reproducible and reliable with a detection limit of 7.5 ng/ml in biological fluids. The mean coefficients of variation for concentrations within the range of the standard curve range were 3.84 and 2.56%, respectively, for intra-day analysis and 4.48 and 4.26%, respectively, for inter-day analysis. Application of the development was successfully demonstrated by quantifying the concentration of I in both dog and rat plasma samples following an intravenous or oral dose of 5 mg/kg I.


Gas chromatographic-mass spectrometric determination of 11-dehydrothromboxane B2 in human urine.

The extension of a method for the determination of thromboxane B2 (TxB2), 2,3-dinor-TxB2, 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and 2,3-dinor-6-keto-PGF1 alpha to quantify 11-dehydro-TxB2 in the same urinary sample is described. After phenylboronic acid and C18 column chromatography, 11-dehydro-TxB2, which is present in urine as the lactone and its corresponding hydroxy acid, was quantitatively converted into its lactone form for a thin-layer purification step and pentafluorobenzyl esterification. Quantification of eicosanoids was achieved by analysing their trimethylsilyl ethers with gas chromatography and negative-ion chemical ionization mass spectrometry. The overall recovery from urine for tritiated 11-dehydro-TxB2 was 80%. The detection limit was 10 pg/ml. The method was applied to the determination of these eicosanoids in volunteers and in patients suffering from acute myocardial infarction.


High-performance liquid chromatographic method for the quantitation of bupivacaine, 2,6-pipecoloxylidide and 4'-hydroxybupivacaine in plasma and urine.

A high-performance liquid chromatographic method with ultraviolet detection at 210 nm for quantitation of bupivacaine and two of its metabolites from plasma and urine is described. The compounds are extracted into n-hexane-isopropanol (5:1), evaporated and the reconstituted residue injected onto a reversed phase C18 column. Standard curves for all compounds were linear (r2 greater than 0.999) in the range 20-2000 ng/ml, with a limit of detection of 10 ng/ml. The inter-day coefficients of variation ranged between 2.7 and 12.2%. The method was applied to analyse bupivacaine and metabolite concentrations in patients on long-term epidural bupivacaine-fentanyl infusions.


Analysis of terbutaline in human plasma by high-performance liquid chromatography with electrochemical detection using a micro-electrochemical flow cell.

A high-performance liquid chromatographic method is described for the determination of terbutaline in human plasma in the range 1-35 ng/ml. Detection was achieved using a carbon fibre micro-electrochemical detector and a column-switching system. The microelectrode cell has advantages over conventional glassy carbon electrode-based detection systems in that it is easy to prepare, flexible in its operation and suffers less trouble from problems such as air bubbles and leaks. Furthermore, it has a better detection limit for terbutaline (0.8 ng/ml) to that obtained using a conventional glassy carbon electrode flow detector (2 ng/ml). Sample clean-up was by on-line solid-phase extraction with column switching, providing a method which was sensitive and reproducible, where the mean overall coefficient of variation was 5.60% and drug recovery in excess of 86% at the concentration levels studied.


Determination of lansoprazole and its metabolites in plasma by high-performance liquid chromatography using a loop column.

A high-performance liquid chromatographic method for the simultaneous determination of lansoprazole, a new proton pump inhibitor, and its metabolites in human plasma is described. Lansoprazole, its metabolites and an internal standard were extracted with tert.-butyl methyl ether. Samples were injected using an automatic injector via a loop column, and separation was obtained using a reversed-phase column under isocratic conditions. The absorbance was monitored at 285 and 303 nm. The quantification limit was 2 ng/ml for lansoprazole and 3 or 5 ng/ml for the metabolites. No endogenous compounds were found to interfere. The mean overall recovery was between 75 and 95% for lansoprazole and its metabolites. This method is suitable for pharmacokinetic studies.


Determination of brodimoprim and its hydroxy metabolite in human plasma, blood and urine by high-performance liquid chromatography.

A high-performance liquid chromatographic method was developed to measure the concentration of brodimoprim and its metabolite, hydroxybrodimoprim, in small volumes of blood, plasma and urine. The procedure involved a simple extraction step with chloroform, followed by chromatographic separation on a short reversed-phase column deactivated for the analysis of basic compounds. The column effluent was monitored by fluorescence (excitation wavelength 290 nm, emission wavelength 340 nm). The recoveries of both compounds were similar in all three biological fluids, and averaged 84 and 72%, respectively. The detection limit for both compounds reached 5 ng/ml. No endogenous compound interfered in the assay. The linearity of the method and its within- and between-day precision were analytically satisfactory.


Separation of theophylline and its analogues by micellar electrokinetic chromatography: application to the determination of theophylline in human plasma.

Micellar electrokinetic chromatographic separation of theophylline and its analogues was investigated using sodium dodecyl sulphate (SDS) as a micellar phase. The effects of pH, micelle concentration, applied voltage and temperature on the separation and preliminary quantitative analysis were studied for the determination of theophylline in human plasma. The data indicate that this technique could be used as the reference or routine method of theophylline measurement in therapeutic drug monitoring.


Nucleotide preparation from cells and determination of nucleotides by ion-pair high-performance liquid chromatography.

Procedures for the analysis of cellular purine and pyrimidine nucleotides are described. The commonly used perchloric acid and especially the trichloroacetic acid methods for nucleotide extraction interfere with ion-pair high-performance liquid chromatography, but we have developed such a system for the separation and determination of major cellular nucleotides in biological matrices, including tri-, di-, monophosphates, cAMP, cGMP, NAD, NADP, UDP-glucose and UDP-galactose. Compared with perchloric acid extraction, no degradation of the nucleotide standards used was observed with respect to triphosphates and other relatively unstable nucleotides. Cellular nucleotides were extracted by lysing cells in a hypotonic buffer containing an ion-pair reagent (tetrabutylammonium hydrogen-sulphate) to decrease enzymic degradation of nucleotides in combination with ultrafiltration of the cell lysate to remove compounds of higher molecular mass, for example enzymes. This method is a simple and reproducible procedure for investigating nucleotide pools in cells.


Improved and simplified tissue extraction method for quantitating long-chain acyl-coenzyme A thioesters with picomolar detection using high-performance liquid chromatography.

A method has been developed that permits rapid and easy tissue extraction of long-chain acyl-coenzyme A (acyl-CoA) thioesters with sensitive quantitation by reversed-phase high-performance liquid chromatography (RP-HPLC). Tissue homogenants are extracted using a reserve Bligh-Dyer technique, and long-chain acyl-CoA esters are harvested in the methanolic aqueous phase. Complex lipids and phospholipids are removed in the chloroform-rich organic Bligh-Dyer second phase, and long-chain acyl-CoA compounds are further purified from the methanolic aqueous Bligh-Dyer first phase on C18 extraction columns after removal of the methanol. The eluted and purified acyl-CoA esters are then quantitated by RP-HPLC using heptadecanoyl-CoA as an internal standard resulting in a detector sensitivity of about 12 pmol. Ten long-chain acyl-CoA esters from C12:0 to C20:4 were identified and separated from canine renal cortex and murine liver samples. The predominant acyl-CoA peaks from both kidney and liver were 14:0, 16:1, 16:0, 18:1, 18:2 and 20:4. Murine liver also produced 18:0 and all peaks disappeared after alkaline hydrolysis of the samples. This extraction and quantitation technique can successfully be used for tissue samples as small as 20 mg, and many samples can be processed in a short period of time. The simplicity of the extraction procedure and the sensitivity of the assay make this an attractive alternative approach to quantitating long-chain acyl-CoA thioesters from complex biological samples such as tissues.


Determination of retinyl palmitate and total vitamin A content in liver and liver-based ready-to-eat foods.

The determination of retinyl palmitate and total vitamin A in liver and liver-based ready-to-eat foods is described. The method is very simple as sample preparation is minimal, and the isocratic elution of the C18 column with pure methanol does not necessitate a sophisticated instrumental set-up. The method is accurate with high recoveries (100.6 +/- 9.3%, mean +/- S.D., n = 23), and precise with within-day and between-day coefficients of variation of less than 5.5% (n = 13) and less than 16% (n = 6), respectively.


Simultaneous determination of nitroglycerin and its dinitrate metabolites by capillary gas chromatography with electron-capture detection.

A major problem in the analysis of nitroglycerin (propanetriol trinitrate, GTN) and its metabolites [propanetriol 1,2-dinitrate (1,2-GDN) and propanetriol 1,3-dinitrate (1,3-GDN)] has been the adsorption of the nitro compounds to glass apparatus during sample preparation or injection. In this paper we present a method determining GTN, 1,2-GDN and 1,3-GDN in plasma at low nanomolar level using capillary gas chromatography with electron-capture detection. The detection limits are about 0.2 nM in plasma. Adsorption of the nitro compounds to glassware is prevented by using triethylamine, giving a simple sample preparation and accurate results. Thus, the laborious and time-consuming silanization of the glassware is avoided.


Gas chromatographic determination of meprobamate in serum or plasma after solid-phase extraction.

This gas chromatographic technique of determining meprobamate is based on a solid-phase extraction permitting a time reduction of the analysis and improving sensitivity. Quantification is realized on 500 microliters of plasma. The method uses etidocaine as internal standard and does not require derivatization. Thus it is simple, rapid, sensitive and applicable in forensic and clinical toxicological laboratories.


Automated high-performance liquid chromatographic method for the simultaneous determination of cefotiam and delta 3-cefotiam in human plasma using column switching.

An automated high-performance liquid chromatographic method using column switching was established for the simultaneous determination of cefotiam (I) and delta 3-cefotiam (II) in human plasma after oral administration of cefotiam hexetil dihydrochloride. The method allowed the determination of analytes in plasma by the direct injection of diluted specimen with phosphate buffer. The analytes were enriched onto the C18 short pretreatment column by 0.05 M phosphate buffer (pH 7.7), while proteins and endogenous hydrophilic substances in plasma were washed off to waste. The enriched analytes were then back-flushed onto the analytical C18 column, separated by a mixture of 0.05 M phosphate buffer (pH 7.7)-acetonitrile (88:12, v/v) and detected by the ultraviolet absorbance at 254 nm. Recoveries from spiked plasma were quantitative, and the coefficients of variation were below 4%. The lower detection limits in plasma were 10 ng/ml for both I and II. Concentrations of I and II in plasma determined by the present method were in good agreement with those obtained by the conventional deproteinization method.


High-performance liquid chromatographic determination of 3,5-dimethylhippuric acid in the occupational exposure to trimethylbenzenes.

A high-performance liquid chromatographic method for the measurement of urinary 3,5-dimethylhippuric acid (3,5-DMHA) in the human biological monitoring of the occupational exposure to trimethylbenzenes has been developed. 3,5-DMHA was extracted from urine with ethyl acetate. The organic phase was dried under vacuum and the resultant product, dissolved in the mobile phase, was analysed by an isocratic system and a programmable photodiode-array detector adjusted to 205 nm. The mobile phase was water-acetonitrile (80:20, v/v) containing 0.1% acetic acid. 3,5-DMHA was chromatographed on a reversed-phase Supelco C18 column (3 microns; 15 cm x 0.46 cm I.D.), and identified by its retention time and ultraviolet spectrum. Quantitation was performed by peak area. The detection limit of the method is 30 ng/ml and the recovery and the accuracy are 96%.


Extractionless determination of diclofenac sodium in serum using reversed-phase high-performance liquid chromatography with fluorimetric detection.

The author describes a method of using reversed-phase high-performance liquid chromatography with fluorimetric detection for the assay of diclofenac sodium in serum. The method is sensitive down to 20 ng/ml (250-microliters loop). Elution is at pH 6.2 with methanol in 0.05 M phosphate buffer (43:57, v/v) on a 25-cm Spherisorb S5 ODS2 column. Detection is at an excitation wavelength of 282 nm and an emission wavelength of 365 nm. Serum sample size is 100 microliters. Sample protein, to which diclofenac is highly bound, is first denatured by heat and then with methanol to release the diclofenac prior to centrifugation and injection of 100 microliters (or 250 microliters) of the clear supernatant. Harmol, with similar fluorescence and polarity characteristics to diclofenac, is a satisfactory internal standard. At the 1 micrograms/ml level intra-sample reproducibility is better than 2%, whilst inter-sample reproducibility is 4.6%. Detector response is linear from 40 ng/ml to 20 micrograms/ml (100-microliters loop).


Method for the quantitation of iodothyronines in body tissues and fluids using high-performance liquid chromatography.

The separation and quantitation of iodotyrosines and iodothyronines [3-monoiodo-L-tyrosine, 3,5-diiodo-L-tyrosine, 3,5-, 3,3' and 3',5'-diiodo-L-tyronines, 3,5,3'-triiodo-L-thyronine (T3), reverse 3,3',5'-triiodo-L-thyronine and 3,3',5,5'-tetraiodo-L-thyronine (T4)] from animal tissues (brain, liver and serum) by a new high-performance liquid chromatographic (HPLC) method is described. Rats were infused with iso-osmotic sodium chloride containing 100 microM phloretin to block deiodination. The tissues were extracted using differential pH values to separate other amines from the amine containing iodothyroid hormones. Aliquots of tissue extracts (25-100 microliters) were reacted overnight with 5-dimethylaminonaphthalene-1-sulfonyl chloride and their iodotyrosine and iodothyronine content determined by HPLC utilizing fluorimetric detection. Resolution of the individual compound peaks was achieved by gradient elution with a 3.0 mM H3PO4 buffer. Greater sensitivity has been achieved (less than 1.0 pmol/g) utilizing fluorescence rather than ultraviolet absorbance for the quantitation of these iodinated compounds. The method is superior also to other methods in that recoveries, based on those of 125I-labelled T4 and T3, were 89-97%.


Sensitive assay for triazolam in plasma following low oral doses.

At low doses of triazolam currently recommended increased assay sensitivity is required for measurement of low plasma concentrations. A highly sensitive capillary gas chromatographic analytical method with a limit of detection of 0.02 ng/ml was developed and used to describe the pharmacokinetics of triazolam following the oral intake of 0.125, 0.250 and 0.375 mg. Six male subjects were studied with blood sampling at the following times: 0, 15, 30 and 45 min and 1, 1.5, 2.0, 2.5, 3, 4, 5, 6 and 8 h. The mean pharmacokinetic parameters for the three doses, respectively, were as follows: half-life, 2.7 +/- 0.4, 3.2 +/- 0.5 and 3.2 +/- 0.6 h; apparent oral clearance, 302.3 +/- 59.0, 260.2 +/- 67.9 and 328.6 +/- 77.8 ml/min; apparent volume of distribution, 64.3 +/- 9.6, 62.0 +/- 12.6 and 73.3 +/- 7.7 l; time to maximum concentration, 0.7 +/- 0.2, 0.6 +/- 0.1 and 0.8 +/- 0.3 h; maximum concentration, 2.2 +/- 0.3, 4.3 +/- 0.6 and 5.0 +/- 0.5 ng/ml; and the area under the concentration-time curve (AUC) up to 8 h, 6.8 +/- 1.2, 16.8 +/- 2.9 and 19.6 +/- 3.5 ng/ml h; and AUC extrapolated to infinity, 8.5 +/- 1.7, 21.4 +/- 4.4 and 26.3 +/- 7.2 ng/ml h. There were no significant differences in the half-life, clearance, volume of distribution and time to maximum concentration among the three doses. The AUC was significantly different on the three occasions and was linearly correlated with dose: r = 0.64 (p less than 0.005).


Simple and high-yield purification of urine protein 1 using immunoaffinity chromatography: evidence for the identity of urine protein 1 and human Clara cell 10-kilodalton protein.

A simple and high-yield purification procedure for urine protein 1 (UP1) using anti-UP1 immunoaffinity chromatography is described. Pure UP1 was obtained in a final yield of 60.2%, and observed as a single electrophoretic band and as a single peak on reversed-phase high-performance liquid chromatography. The N-terminal amino acid residue of UP1 was found to be glutamic acid, contrary to what was reported previously. Furthermore, the N-terminal sequence of UP1 up to 53 amino acids was confirmed to be identical with that of mature human Clara cell 10-kilodalton protein, which inhibits phospholipase A2 activity.


Determination of nicotinamide-adenine dinucleotide and thiazole-4-carboxamide-adenine dinucleotide in human leukocytes by reversed-phase high-performance liquid chromatography.

A high-performance liquid chromatographic assay for cellular nicotinamide-adenine dinucleotide and thiazole-4-carboxamide-adenine dinucleotide is presented that is appropriate for analysis of these dinucleotides in extracts of Ficoll-purified human leukemic cells. The separation, which is effected by reversed-phase chromatography, is highly reproducible and the limit of quantitation is as low as 10-15 pmol. The stability of these compounds in neutralized perchloric acid extracts is addressed and the applicability of the procedure to clinical specimens is demonstrated.


pH titration curves of the desialylated human alpha 1-acid glycoprotein variants by combined isoelectrofocusing-electrophoresis: utilization in the development of a fractionation method for the protein variants by chromatography on immobilized metal affinity adsorbent.

Using a two-dimensional isoelectrofocusing (IEF)-electrophoresis technique, the pH titration curves of the three main desialylated variants (F1, S and A) of human alpha 1-acid glycoprotein (AAG) were studied to assist in the development of a fractionation method for the AAG variants. For this purpose, different AAG samples, each corresponding to one of the three main phenotypes of the protein (F1S/A, F1/A and S/A), were first purified by chromatographic separation of individual human plasma samples on immobilized Cibacron Blue F3G-A. The purified AAG samples were then disialylated and their heterogeneity was checked by analytical IEF. The pH-mobility curves of the desialylated AAG samples were displayed in polyacrylamide gel slabs, under a constant set of experimental conditions, by carrying out electrophoresis of the protein samples perpendicularly to two stationary pH gradients: a large gradient (pH 3.5-9.5) and a narrow gradient (pH 5-8). The curves showed that all the desialylated variants of AAG exhibited small charge differences and moved closely together between about pH 3.5-5.5 and pH 7.5-9.5. However, the variants were found to show microheterogeneity in their total charge between about pH 5.5 and 7.5 due to the titrated ionizable groups involved along this pH zone. This microheterogeneity was assumed to be accounted for by the existence of differences between the titratable histidyl residues of the AAG variants. Consequently, the interactions of the variants with immobilized transition metal ions were studied at pH 7, using affinity chromatography on an iminodiacetate Sepharose-Cu(II) gel. It was found that the A variant was strongly bound by immobilized Cu(II) ions, whereas the F1 and S variants interacted non-specifically with the immobilized ligand. This finding allowed the development of a rapid and effective fractionation method for desialylated AAG into its A and F1 or S variants, depending on the AAG phenotype, by chromatography on an immobilized affinity Cu(II) adsorbent. The quantitative relationships between immobilized Cu(II) ions and desialylated AAG (the apparent association constant and gel protein-binding capacity) were also determined using a non-chromatographic equilibrium binding technique.


Solid-phase extraction with supercritical fluid elution as a sample preparation technique for the ultratrace analysis of flavone in blood plasma.

A new sample preparation technique, solid-phase extraction with supercritical fluid elution, was developed for the selective isolation of ultratrace levels of drugs from plasma. Plasma samples spiked with a drug were applied to octadecylsilane cartridges and the cartridges were then washed, briefly dried and directly fitted into cells for subsequent supercritical fluid elution. The absolute recovery was studied by using a radiolabeled model compound. The extraction selectivity was examined by chromatographing the extracts with a reversed-phase high-performance liquid chromatographic method with ultraviolet detection. The effects of extraction pressure and the length of capillary restrictors on drug recovery were examined in order to determine the optimal conditions for supercritical fluid elution. The performance of the method was compared to that of conventional solid-phase extraction in terms of recovery, selectivity, precision and accuracy of analysis. Flavone was used as the model compound and dog plasma as the biological matrix for these studies.


Detection of benzo[a]pyrene sulfate and glucuronide conjugates in cell culture medium by directly coupled microbore high-performance liquid chromatography-fast atom bombardment mass spectrometry.

An improved method is described for detecting glucuronide and sulfate conjugates of benzo[a]pyrene in medium from cell cultures treated with benzo[a]pyrene. This method is based on a microbore high-performance liquid chromatograph directly coupled to a high-resolution continuous-flow fast atom bombardment mass spectrometer. Sulfate and glucuronide conjugates, as well as some structural isomers of glucuronide conjugates, were fully separated by the reversed-phase microbore high-performance liquid chromatography conditions used in this study. Since the method does not rely on the use of radiolabeled materials, it may be used to detect conjugates of a wide variety of hydrocarbons. The high sensitivity and selectivity of the method were demonstrated by detecting conjugates in the media of cell cultures derived from mice, hamsters and humans.


Determination of tannic acid and its phenolic metabolites in biological fluids by high-performance liquid chromatography.

A method for the identification and determination of tannic acid and its phenolic metabolites in biological fluids by high-performance liquid chromatography was developed. Tannic acid and four phenolic compounds, namely gallic acid, pyrogallol, 4-O-methylgallic acid and ellagic acid, were successfully extracted from the biological fluids by using ethyl acetate at acidic conditions. Gallic acid, pyrogallol and 4-O-methylgallic acid were found in the sheep urine, gallic acid, 4-O-methylgallic acid and ellagic acid in plasma, and gallic acid and ellagic acid in abomasal fluid after abomasal dosing of tannic acid. Tannic acid was found in the plasma apart from the abomasal fluid into which it was administered. The concentrations of tannic acid, gallic acid, pyrogallol, 4-O-methylgallic acid and ellagic acid in plasma, abomasal fluid and urine were measured. This method could be applied to measurement of other hydrolysable tannins and their phenolic metabolites in biological materials.


Validation of the simultaneous determination of methylprednisolone and methylprednisolone acetate in human plasma by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic procedure was developed that accurately quantitates methylprednisolone (MP) and methylprednisolone acetate (MPA) in human plasma over the range 2.00-50.0 ng/ml. The internal standard, fluorometholone, was added to an aliquot of sodium fluoride-potassium oxalate-derived plasma. Samples were prewashed with hexane and extracted twice with methylene chloride. The extracts were dried with anhydrous sodium sulfate, centrifuged, and the organic layer separated and dried under nitrogen. The samples were reconstituted in mobile phase and washed an additional time with hexane before 100 microliters were injected onto a Beckman/Altex Ultrasphere ODS column with ultraviolet absorbance detection at 254 nm. Composition of the mobile phase was acetonitrile-water-glacial acetic acid (33:62:5, v/v/v). Calibration curves were obtained by unweighted, linear regression of peak-height ratios of MP (or MPA)/internal standard versus theoretical concentrations of MP or MPA using a Hewlett-Packard 3357 Laboratory Automation System. Extraction efficiencies for MP and MPA over the linear range were 86.4 and 84.7%, respectively. This method was successfully implemented for the analysis of specimens generated from a single-dose bioavailability and safety study for a new formulation of Depo-Medrol sterile aqueous suspension.


High-performance liquid chromatographic separation of malondialdehyde-thiobarbituric acid adduct in biological materials (plasma and human cells) using a commercially available reagent.

The assay of malondialdehyde (MDA) is widely used in clinical chemistry laboratories to investigate lipid peroxidation in oxidative pathologies. In the present work, the thiobarbituric acid (TBA) reaction was carried out on plasma, human erythrocytes and fibroblasts. The reagents used were those of the fluorimetry MDA kit manufactured by Sobioda. We have defined the application of this kit to high-performance liquid chromatography. This adaptation satisfied the criteria of good analytical practice. The detection limit was 2.5 pmol per injection. The retention time of the MDA-TBA2 peak (4.96 +/- 0.07 min) led to excellent resolution of the complex. The within-assay (6-12%) and between-assay (11-12%) precisions were satisfactory. The analytical recovery of MDA after spiking samples of human plasma with tetraethoxypropane standards varied from 70 to 100%. The mean lipoperoxide concentration determined in 32 healthy adults (20-40 years) was 1.04 +/- 0.23 mumol l-1 in plasma. Applied to the erythrocytes of fifteen laboratory workers, the method furnished physiological values of 0.59 +/- 0.21 mumol l-1. Concentrations were significantly higher in chronic renal dialysis patients (4.15 +/- 2.35 mumol l-1. The MDA content of fibroblasts cultured in standard medium was 0.38 +/- 0.04 mumol per g of protein and increased (5.78 +/- 1.38 mumol per g of protein) if the cells were grown in an iron-enriched medium. This accurate high-performance liquid chromatographic method for detection of MDA is the first one which can be applied to plasma, red blood cells and cultured cells. This technique will prevent false positives and should make inter-laboratory comparisons possible.


Determination of enantiomers of 1-benzyl-4-[(5,6-dimethoxy-1-indanon)-2-yl]methylpiperidine hydrochloride (E2020), a centrally acting acetylcholine esterase inhibitor, in plasma by liquid chromatography with fluorometric detection.

Two high-performance liquid chromatographic methods for the determination of enantiomers of E2020, 1-benzyl-4-[(5,6-dimethoxy-1-indanon)2-yl]methylpiperidine (I) hydrochloride, in rat plasma have been developed. The first method involves chiral separation of I on an ovomucoid-bonded column, and native fluorescence detection of I with excitation at 318 nm and emission at 390 nm. The fluorometric detection is without interference from background components and is about five times more sensitive than ultraviolet detection at 271 nm. The method was successfully applied to monitoring the racemization of each enantiomer of I in buffer solutions and in rat plasma. The second method involves separation of I from background components of rat plasma on an achiral column, collection of the I fraction into a sample loop, concentration or I to a trap column, transfer of I to a chiral column, resolution of the enantiomers of I on the chiral column and fluorometric detection of the enantiomers of I with excitation at 318 nm and emission at 390 nm. The detection limits of I and each enantiomer of I were 1 and 1 ng/ml, respectively, with a 200-microliters injection of deproteinized plasma samples.


Detection of methandienone (methandrostenolone) and metabolites in horse urine by gas chromatography-mass spectrometry.

The metabolic transformation of methandienone (I) in the horse was investigated. After administration of a commercial drug preparation to a female horse (0.5 mg/kg), urine samples were collected up to 96 h and processed without enzymic hydrolysis. Extraction was performed by a series of solid-liquid and liquid-liquid extractions, thus avoiding laborious purification techniques. For analysis by gas chromatography-mass spectrometry, the extracts were trimethylsilylated. Besides the parent compound I and its C-17 epimer II, three monohydroxylated metabolites were identified: 6 beta-hydroxymethandienone (III), its C-17 epimer (IV) and 16 beta-hydroxymethandienone (V). In addition, three isomers of 6 beta,16-dihydroxymethandienone (VIa-c) were discovered. Apparently, reduction of the delta 4 double bond of 16 beta-hydroxymethandienone (V) in the horse yields 16 beta,17 beta-dihydroxy-17 alpha-methyl-5 beta-androst-1-en-3-one (VII). Reduction of the isomers VIa-c results in the corresponding 6 beta,16,17-trihydroxy-17-methyl-5 beta-androst-1-en-3-ones (VIIIa-c). The data presented here suggest that screening for the isomers of VI and VIII, applying the selected-ion monitoring technique, will be the most successful way of proving methandienone administration to a horse.


Identification of urinary acylcarnitines using gas chromatography-mass spectrometry: preliminary clinical applications.

Many disorders of organic acid metabolism are associated with abnormalities in the levels of acylcarnitines excreted in urine. Profiling of urinary acylcarnitines allows diagnosis and characterisation of many acidurias and acidemias, monitoring dietary treatment of such patients, and elucidation of the metabolism of some exogenous acidic compounds. Urine (ca. 0.5 ml) was subjected to a simple work-up by ion-exchange chromatography, and the isolated acylcarnitines were derivatized by cyclization in 35 min to give volatile lactones that are compatible with gas chromatography-mass spectrometry using electron or chemical ionization. The feasibility of this new and affordable procedure has been confirmed by identifying urinary acylcarnitines in cases of medium-chain acyl-coenzyme A dehydrogenase deficiency, propionic acidemia and isovaleric acidemia.


Simple and sensitive determination of diacetyl and acetoin in biological samples and alcoholic drinks by gas chromatography with electron-capture detection.

Acetoin was quantitatively oxidized into diacetyl by Fe3+ in 1 M perchloric acid. The reaction of diacetyl with 4,5-dichloro-1,2-diaminobenzene afforded 6,7-dichloro-2,3-dimethylquinoxaline (DCDMQ), which was extracted by benzene containing aldrin (25 ng/ml) as an internal standard, and determined by gas chromatography with electron-capture detection. The method is very simple and sensitive. The detection limit of DCDMQ (either diacetyl or acetoin) was 10 fmol/microliters of the benzene extract, and the determination limit of DCDMQ (either diacetyl or acetoin) was 50 fmol/microliters of the extract. Both acetoin and diacetyl could be determined in 0.1 ml of normal human urine or blood, and both were found in rat liver, kidney and brain. The method was also applied to the determination of acetoin and diacetyl in alcoholic drinks.


Thermospray liquid chromatography-mass spectrometry of corticosteroids.

A high-performance liquid chromatographic method was developed for thermospray mass spectrometric analysis of steroidal hormones. Using a Nova-Pak C18 reversed-phase column and isocratic elution with a solvent comprised of 25 mM ammonium formate in 30% acetonitrile, corticosteroids were separated within 10 min. This solvent also permitted ultraviolet absorbance detection down to 220 nm with low-nanogram sensitivity. The use of acetonitrile was favourable for thermospray mass spectrometric analysis because mass spectra were obtained with a pseudomolecular ion as the base peak. A combination of liquid chromatography, ultraviolet absorbance detection and thermospray mass spectrometry provided a sensitive and reliable method for unequivocal confirmation of the presence of steroidal drugs in equine urine.


Determination of conjugated bile acids in human urine by high-performance liquid chromatography with chemiluminescence detection.

A qualitative and quantitative analysis of the conjugated 1 beta- and 6 alpha-hydroxy bile acids, including common bile acids, in human urine using high-performance liquid chromatography with chemiluminescence detection is described. After extraction of urine with C18 silica cartridges, the bile acids were separated into non-conjugated, glycine, taurine and sulphate fractions by ion-exchange chromatography on a lipophilic gel. Solvolysis of the sulphate was carried out by treatment with trifluoroethanol in acetone containing hydrochloric acid, and the liberated amino acid conjugates were fractionated again. The individual bile acids were separated on a reversed-phase C18 column (Bile Pak II), with detection by an immobilized 3 alpha-hydroxysteroid dehydrogenase enzyme reactor and chemiluminescence reaction of the generated NADH using 1-methoxy-5-methylphenazinium methylsulphate-isoluminol-microperoxidase system. The assay method showed the detection limits ranging from 8 to 250 pmol for the bile acids tested. Analysis of urine samples obtained from newborns, non-pregnant women and women in late pregnancy showed a large difference in bile acid composition and conjugation mode, suggesting that bile acid metabolism is different during fetal and neonatal periods.


Use of biospecific interactions of collagen, fibronectin and their fragments in affinity chromatography.

Various aspects of the application of fibronectin-collagen biospecific interactions in affinity chromatography are described. A new biospecific method for one-stage isolation of collagen peptides containing fibronectin-binding sites is proposed. The alpha 1 CB7-peptide of type-I collagen cyanogen bromide cleavage was isolated by means of affinity chromatography on adsorbents containing an immobilized gelatin-binding domain (45,000 relative molecular mass) of fibronectin. The method gives highly purified preparations of alpha 1 CB7-peptide. This peptide, as well as some other collagen molecular fragments (alpha-chains, beta-components, alpha 1 CB8-peptide), were immobilized on Sepharose, and the properties of such affinity adsorbents obtained were studied. Adsorbents with immobilized alpha-chains and alpha 1 CB7-peptide had a fibronectin-binding capacity 1.5-2.0 times higher than commercial gelatin-Sepharose. Large-scale production of highly purified fibronectin from human plasma, using affinity chromatography on immobilized individual alpha-chains of collagen, was developed.


Use of a carrier for quantitation of a new dihydropyridine calcium antagonist (OPC-13340) in human plasma by highly sensitive gas chromatography with negative-ion chemical ionization mass spectrometry.

A sensitive gas chromatographic-mass spectrometric method for the quantitation of (+/-)-methyl 3-phenyl-2(E)-propenyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate (OPC-13340, I), a new dihydropyridine calcium antagonist with a potent and long-acting antihypertensive and antianginal effect, was developed in order to elucidate its pharmacokinetics. Dihydropyridine calcium antagonist have been usually quantifed by this technique in the negative-ion chemical ionization mode. However, direct application of this method to quantify trace amounts of I in biological fluids completely failed, owing to its adsorption on the column and oxidation of its dihydropyridine ring. Human plasma containing I and (+/-)-[2H3]methyl 3-phenyl-2(E)-propenyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate (II), the internal standard, was extracted with n-hexane-diethyl ether under weakly basic conditions (pH 8). In order to prevent adsorption of the compounds on the column, (+/-)-[2H5]ethyl 3-phenyl-2(E)-propenyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate (III), an analogue of I, was added to the extracts as a carrier. In addition, this carrier was also effective in preventing the oxidation of I. The quantitation limit of I in human plasma by this method was found to be less than 30 pg/ml. Thus, the method is sufficiently sensitive to study the pharmacokinetics of I in humans.


Nanogram level quantitation of oxycodone in human plasma by capillary gas chromatography using nitrogen-phosphorus selective detection.

A sensitive and specific capillary gas chromatographic assay is reported for the quantitation of oxycodone in human plasma. The technique involves a single extraction of oxycodone and internal standard (hydrocodone) from plasma by toluene containing 1% isopropanol. Separation is achieved on a methyl silicone (HP-1) fused-silica capillary column (25 m x 0.2 mm I.D., 0.33 microns film thickness) and detection is by nitrogen-phosphorus selective mode. The minimum quantifiable limit is 1.8 ng/ml using 2 ml of plasma. The method is applicable to characterize the plasma profile of oxycodone in humans after a single oral 5-mg oxycodone hydrochloride tablet.


Gas chromatographic headspace analysis of sevoflurane in blood.

We have developed a rapid, simple and precise gas chromatographic headspace analysis for sevoflurane in blood which circumvents problems associated with the high volatility and low blood/gas partition coefficient of this anesthetic drug. Blood standards are easily prepared by volumetric addition of a saturated aqueous solution of sevoflurane. Likewise, internal standardization is achieved using a saturated aqueous solution of halothane. Chromatographic conditions are similar to those commonly used for the analysis of blood ethanol. A simple method is also described for the preparation of stable and precise, aliquots of quality control materials for this assay.


Allosteric and competitive displacement of drugs from human serum albumin by octanoic acid, as revealed by high-performance liquid affinity chromatography, on a human serum albumin-based stationary phase.

A chiral stationary phase for high-performance liquid chromatography, based upon immobilized human serum albumin (HSA), was used to investigate the effect of octanoic acid on the simultaneous binding of a series of drugs to albumin. Octanoic acid was found to bind with high affinity to a primary binding site, which in turn induced an allosteric change in the region of drug binding Site II, resulting in the displacement of compounds binding there. Approximately 80% of the binding of suprofen and ketoprofen to HSA was accounted for by binding at Site II. Octanoic acid was found to also bind to a secondary site on HSA, with much lower affinity. This secondary site appeared to be the warfarin-azapropazone binding area (drug binding Site I), as both warfarin and phenylbutazone were displaced in a competitive manner by high levels of octanoic acid. The enantioselective binding to HSA exhibited by warfarin, suprofen and ketoprofen was found to be due to differential binding of the enantiomers at Site I; the primary binding site for suprofen and ketoprofen was not enantioselective.


Stereospecific high-performance liquid chromatographic assay of pirprofen enantiomers in rat plasma and urine.

A stereospecific high-performance liquid chromatographic method was developed for the assay of pirprofen enantiomers in rat plasma and urine. Following addition of internal standard (ketoprofen) and acidifier (L-ascorbic acid) to biological fluids, pirprofen was extracted into an isopropanol-isooctane (5:95) mixture. Diastereomers of pirprofen enantiomers, which were formed using L-leucinamide, were separated on a reversed-phase column with ultraviolet detection at 275 nm using 0.06 M KH2PO4-acetonitrile-triethylamine (64:36:0.1) as mobile phase. The limit of quantitation was 0.1 microgram/ml for each enantiomer, based on 100 microliters of rat plasma. No spontaneous oxidation of pirprofen to its pyrrole metabolite occurred during sample preparation and analysis. In three female rats which were dosed with 10 mg/kg racemic pirprofen orally, plasma concentrations of the enantiomers could be followed for 24 h. Pirprofen enantiomers in plasma were virtually unconjugated, and negligible concentrations of pyrrole metabolites were observed. Less than 10% of the total dose was recovered in urine as intact drug and its glucuroconjugates. The assay was found suitable for the study of the pharmacokinetics of pirprofen enantiomers in the rat.


Simultaneous quantitative determination of naproxen, its metabolite 6-O-desmethylnaproxen and their five conjugates in plasma and urine samples by high-performance liquid chromatography on dynamically modified silica.

The glucuronides of the anti-inflammatory drug naproxen and its metabolite 6-O-desmethylnaproxen have been produced on a preparative scale by enzymatic synthesis. 6-O-Desmethylnaproxen, the glycine conjugate of naproxen and the O-sulphate of 6-O-desmethylnaproxen were prepared by chemical synthesis. Naproxen and the purified metabolite and conjugates were used as standards for the analytical investigation of the metabolic pattern of naproxen in humans. A reversed-phase high-performance liquid chromatographic method based on bare silica dynamically modified with cetyltrimethylammonium ions has been developed. The system was optimized to give a separation of naproxen, 6-O-desmethylnaproxen and five conjugates. Using this method it is also possible to deduce the relationship between the amount of the intact ether-glucuronide and acyl-glucuronide of 6-O-desmethylnaproxen.


Fast systematic approach for the determination of drugs in biological fluids by fully automated high-performance liquid chromatography with on-line solid-phase extraction and automated cartridge exchange. Application to cebaracetam in human urine.

A fast stepwise systematic approach for the conversion of conventional reversed-phase high-performance liquid chromatographic (HPLC) assays involving liquid-liquid extraction of biological fluids into fully automated HPLC assays using solid-phase extraction and cartridge exchange is described. The suitability of this procedure is demonstrated for the determination of cebaracetam in human urine.


Comparison of two extraction procedures for urinary organic acids prior to gas chromatography-mass spectrometry.

We have compared a new isolation procedure for urinary organic acids using strong anion-exchange columns with a solvent partition (ethyl acetate) method. Urinary samples from two healthy children and from nine children with organic acidurias were analysed by both procedures. Although the solid-phase extraction is more efficient for polyhydroxy acids, some polar acids, and some glycine derivatives, clinically important compounds such as oxalic, methylcitric, pyruvic, glyoxylic and 2-ketoglutaric acids, are not recovered or are only poorly recovered. However, both procedures may be used as a routine method for the diagnosis of the organic acidurias included in this study.


Analysis of novel imidazoles from isolated perfused rabbit heart by two high-performance liquid chromatographic methods.

The paper reports two analytical high-performance liquid chromatographic methods to detect and quantify cardiac-derived histidyl derivatives. Method A relies on relative hydrophobicities as a basis of separation. Method B is an ion-pairing method in which the compounds are eluted in an entirely different order. Fractions collected from method A have been co-eluted in admixture in method B with authentic reference compounds. Thus the existence of the following imidazole ring-containing compounds derived from heart have been confirmed: N-acetylhistidine, N-acetyl-1-methylhistidine, N-acetylcarnosine, N-acetylhomocarnosine, homocarnosine, anserine, carnosine, balenine. Compounds were found in both tissue samples and perfusates.


Simultaneous determination of cotinine and trans-3'-hydroxycotinine in human serum by high-performance liquid chromatography.

A high-performance liquid chromatographic method with ultraviolet photometric detection has been developed for the quantitation of cotinine and trans-3'-hydroxycotinine in human serum. A solid-phase extraction procedure was performed for the analytes and the internal standard, N-ethylnorcotinine, before chromatography. The use of a 30-cm reversed-phase column and a mobile phase of water-methanol-0.1 M sodium acetate-acetonitrile (67:24.5:6.5:2, v/v), pH 4.3, prevented the co-elution of caffeine with cotinine. The limit of quantitation observed with this method was 5 ng/ml for both cotinine and trans-3'-hydroxycotinine. The present method proved useful for the determination of serum levels of these metabolites, correlating with nicotine daily intake.


Simultaneous determination of (R)- and (S)-naproxen and (R)- and (S)-6-O-desmethylnaproxen by high-performance liquid chromatography on a Chiral-AGP column.

A high-performance liquid chromatographic method for the simultaneous determination of both enantiomers of naproxen and its metabolite 6-O-desmethylnaproxen has been developed. The separation is performed on a column containing alpha 1-acid glycoprotein as the chiral selector. The method has been used for the determination of the enantiomeric purity of the drug substance and the metabolite, and for the simultaneous determination of all four compounds in biological fluids.


High-performance liquid chromatographic method for the determination of mangiferin, likviritin and dihydroquercetin in rat plasma and urine.

The use of reversed-phase high-performance liquid chromatography for the determination of the biologically active plant phenolic compounds mangiferin, likviritin and dihydroquercetin is described. Perchloric acid (35%) was used for deproteinization in the case of mangiferin and likviritin, and acidified methanol for dihydroquercetin. Detection was performed at 254, 275 and 290 nm for mangiferin, likviritin and dihydroquercetin in plasma, and 365, 312 and 290 nm in urine, respectively. The limit of detection was 0.2 micrograms/ml for plasma and 0.5 micrograms/ml for urine.


High-performance liquid chromatographic determination of imidocarb in cattle kidney with cation-exchange clean-up.

A high-performance liquid chromatographic (HPLC) method for the determination of the antiprotozoal agent imidocarb in cattle kidney is developed. The drug is extracted from tissue with acetone in the presence of base. The extract is partitioned between saturated salt and chloroform and the organic layer evaporated to dryness. Clean-up is by cation-exchange solid-phase extraction on a carboxylic acid column. HPLC analysis is carried out on a Spherisorb S3W-PC18 column with ultraviolet detection at 260 nm. Average recoveries at the 0.05 and 0.01 mg kg-1 levels are 77.5 and 76.3%, respectively. The limit of detection is 0.001 mg kg-1.


Determination of picotamide in human plasma and urine by high-performance liquid chromatography.

A high-performance liquid chromatographic method for the determination of picotamide in human plasma and urine is described. After addition of an internal standard (bamifylline), the plasma and urine samples were subjected to liquid-liquid extraction and clean-up procedures. The final extracts were evaporated to dryness and the resulting residues were reconstituted in 100 microliters of methanol-water (50:50, v/v) and chromatographed on a LiChrosorb RP-SELECT B reversed-phase column coupled to an ultraviolet detector monitored at 230 nm. Chromatographic analysis takes about 10 min per sample. The assay was linear over a wide range and has a limit of detection of 0.005 and 0.1 micrograms/ml in plasma and urine, respectively. It was selective for picotamide, accurate and robust and thus suitable for routine assays after therapeutic doses of picotamide.


Rapid determination of propyphenazone in plasma by high-performance liquid chromatography.

A rapid and simple high-performance liquid chromatographic assay for the determination of propyphenazone in plasma is described. Phenylbutazone was used as the internal standard. Plasma proteins were precipitated with acetonitrile before injection onto a 3-microns Supelcosil LC-18 column. The mobile phase, ethanol containing 0.2% (v/v) heptylamine-0.005 M potassium dihydrogenphosphate (30:70, v/v), was used at a flow-rate of 1.3 ml/min. The quantitation was performed by ultraviolet detection at a wavelength of 270 nm. The chromatographic time was 7 min. The within- and between-day coefficients of variation were less than 6% and the recoveries close to 100% for concentrations between 0.4 and 22 mumol/l. The limit of quantitation was 0.4 mumol/l (ca. 100 ng/ml).


Determination of "free" glycerol in human serum reference materials by isotope-dilution gas chromatography-mass spectrometry.

Serum free glycerol analyses are an important part of the preparation and evaluation of human serum reference materials used for the quality assurance of triglyceride assays. However, enzymatic kits for free glycerol analysis obtained from different vendors have, on occasion, provided different results for a given sample. In an effort to establish the "target" glycerol content of selected reference materials, we have established a method for the analysis of serum free glycerol by using isotope-dilution gas chromatography-mass spectrometry, incorporating [1,3-13C2]glycerol as the internal standard. The use of a simplified serum extraction and clean-up procedure resulted in (uncorrected) recoveries of glycerol averaging about 90% before derivatization, and in estimated concentrations for spiked serum pools that corresponded closely to the expected values. A comparison of enzymatic and gas chromatographic-mass spectrometric results for several reference serum pools suggest that the latter method is of value in evaluating and validating routine enzymatic methods for free glycerol analysis.


High-performance liquid chromatographic assay for 1-aminocyclopropanecarboxylic acid from plasma and brain.

A reversed-phase high-performance liquid chromatographic method for the analysis of 1-aminocyclopropanecarboxylic acid (ACPC) from plasma or brain tissue is described. Samples were deproteinized with perchloric acid, centrifuged, alkalinized with potassium hydroxide and recentrifuged. The supernatants were derivatized with o-phthaldialdehyde and injected onto a C18 3-microns column (100 mm x 4 mm I.D.) pumped with 1 ml/min methanol-acetonitrile-0.1 M sodium phosphate buffer pH 6.0 (28:5:67, v/v). The retention times for ACPC and the internal standard were 15 and 31 min, respectively. The minimum detectable amount of ACPC was 0.08 nmol. The extraction recovery of ACPC (2.7-270 nmol) from spiked plasma or brain tissue ranged from 88 to 109%. The intra- and inter-day coefficients of variation for 27 nmol ACPC were 3.9 and 4.9%, respectively. This method was utilized to obtain preliminary pharmacokinetic parameters following ACPC administration to mice.


Endogenous synthesis of N-methylsalsolinol, an analogue of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, in rat brain during in vivo microdialysis with salsolinol, as demonstrated by gas chromatography-mass spectrometry.

N-Methylsalsolinol, an analogue of 1,2,3,6-tetrahydropyridine, is present in the brains of patients with Parkinson's disease. To determine the metabolic pathway for the synthesis of N-Methylsalsolinol in the brain, salsolinol was perfused through the striatum or the substantia nigra of the rat brain by in vivo microdialysis. N-Methylsalsolinol was detected in the brain dialysate samples during microdialysis with salsolinol using gas chromatography-mass spectrometry with selected-ion monitoring. These results demonstrate that endogenous N-methylation of salsolinol into N-methylsalsolinol occurs in the brain in vivo.