Electrospray device for coupling microscale separations and other miniaturized devices with electrospray mass spectrometry.

A miniaturized ion sprayer device is described which is suitable for coupling with chip-based analytical separation devices, multiwell plates, or surfaces containing residues of prepared samples. Two versions of a similar device are described. A "microsprayer" device suitable for coupling to the terminal edge of a capillary electrophoresis (CE) chip is constructed from modified 1/16-in. HPLC fittings. This microsprayer employs a free-standing liquid junction formed via continuous delivery of a flow (2-6 microL/min) of suitable solvent which carries the CE effluent through a pneumatically assisted electrospray (ion spray) needle positioned in front of an atmospheric pressure ionization (API) mass spectrometer. A related but larger "minisprayer" device is also described which employs the same features as the microsprayer, but with an extended sampling capillary tube which can reach into the depths of 96-, 384-, and 1536-multiwell plates containing either sample solutions or dried sample residues. The minisprayer may be positioned in front of an API ion sampling orifice and the multiwell plate positioned stepwise from sample to sample for analysis of trace samples contained in the wells. The resulting infusion-ion spray mass spectrometric analyses can provide sequential analysis of previously prepared biological samples containing small drug compounds, proteins, and related compounds. This same device is also shown to be useful for sampling from a surface containing trace level compounds of biological interest. Results are shown that demonstrate microscale separations and selected ion monitoring (SIM) capillary electrophoresis/mass spectrometry (CE/MS) detection of berberine and palmatine using the microsprayer. SIM ion spray determination of a 2 ng/microL solution of berberine contained as a dry residue in the bottom of a 384-well plate as well as full-scan electrospray mass spectra for low-picomole levels of cytochrome c contained in a 1536-well microtiter plate are shown. The respective micro- and minisprayer devices provide a simple yet effective means of transferring trace-level samples either from a microscale or chip-based separation device as well as samples contained in multiwell plates which are increasingly employed in high-throughput applications in the pharmaceutical industry.

High-throughput bioanalytical LC/MS/MS determination of benzodiazepines in human urine: 1000 samples per 12 hours.

The analytical capabilities of liquid chromatography tandem mass spectrometry for sensitive and highly selective determination of target compounds in complex biological samples makes it well suited for high-throughput analysis. We report the fast separation of six benzodiazepines isolated from human urine via selected reaction monitoring liquid chromatography/mass spectrometry using short dwell times to accommodate fast-eluting chromatographic peaks. The analytes were extracted from human urine samples along with their deuterium-labeled internal standards by a simple liquid-liquid extraction in 96-well plates. Using four autosamplers coupled to one chromatographic column and one tandem mass spectrometer operated in the turbo ion spray mode with positive ion detection, 1152 samples (12 96-well plates) were analyzed in less than 12 h. Through an electronic switching box designed and constructed in-house, the autosamplers were synchronized with the mass spectrometer so that injections were made as soon as the mass spectrometer was ready to collect data. Each run required 30 s to complete with another 7-8 s for the data system to load the next data file to be collected. Chromatographic integrity and ion current response remained relatively constant for the duration of the analyses. The results show acceptable precision and accuracy and demonstrate the feasibility of using fast separations with tandem mass spectrometry for high-throughout analysis of biological samples containing multiple analytes.

Quantitative ionspray liquid chromatographic/tandem mass spectrometric determination of reserpine in equine plasma.

A method based on ionspray liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed for the determination of reserpine in equine plasma. A comparison was made of the isolation of reserpine from plasma by liquid-liquid extraction and by solid-phase extraction. A structural analog, rescinnamine, was used as the internal standard. The reconstituted extracts were analyzed by ionspray LC/MS/MS in the selected reaction monitoring (SRM) mode. The calibration graph for reserpine extracted from equine plasma obtained using liquid-liquid extraction was linear from 10 to 5000 pg ml-1 and that using solid-phase extraction from 100 to 5000 pg ml-1. The lower level of quantitation (LLQ) using liquid-liquid and solid-phase extraction was 50 and 200 pg ml-1, respectively. The lower level of detection for reserpine by LC/MS/MS was 10 pg ml-1. The intra-assay accuracy did not exceed 13% for liquid-liquid and 12% for solid-phase extraction. The recoveries for the LLQ were 68% for liquid-liquid and 58% for solid-phase extraction.

Design and applications of a self-aligning liquid junction-electrospray interface for capillary electrophoresis-mass spectrometry.

A simple self-aligning liquid junction-electrospray interface for coupling a capillary electrophoresis (CE) system to an atmospheric pressure ionization (API) mass spectrometer (CE-MS) was developed. In contrast to previous liquid junction interfaces, the self-aligning liquid junction interface simplifies the precise alignment of the CE capillary and the sprayer needle and uses a positive make-up flow. Several capillary CE-MS applications were run using both the self-aligning liquid junction interface and the widely used sheath flow interface for comparison purposes. The electrospray stability of the self-aligning liquid junction interface is consistently better even when non-volatile electrolyte solutions are used. At first, some band broadening was obtained with the self-aligning liquid junction interface. Experiments with different CE buffer systems suggested that this band broadening was caused by the materials used in constructing the interface. By using a more inert material for the sprayer needle, the self-aligning liquid junction exhibits excellent electrophoretic resolution, comparable sensitivity, and higher signal-to-noise ratios when run under the same conditions as the sheath flow interface.

Relation of mild-to-moderate malnutrition to human development: correlational studies.

The present review focuses on the relation to human behavior and development of anthropometric or dietary indexes of mild-to-moderate malnutrition. The primary goal of the review is to integrate previous research findings with current findings from correlational studies conducted over the past decade. From this integration, the following conclusions may be drawn: 1) Chronic, mild postnatal malnutrition is associated with a variety of cognitive and behavioral deficits across the life span. The role of prenatal malnutrition in this process is less clear. 2) To understand the role chronic mild malnutrition plays in behavior and development, it is necessary to move beyond protein-calorie deficits to consider the role of intake of animal source foods and specific micronutrients such as iron, zinc and B vitamins. 3) Chronic mild malnutrition is embedded in a host of other biological and psychosocial risk factors. As a result, chronic mild malnutrition appears to be a necessary but insufficient condition for producing behavioral deficits. 4) The salience of chronic mild malnutrition as a risk factor is accentuated when other psychosocial-contextual risk factors are also present or when multiple low-level nutrient deficits are interacting. Suggestions for future research directions include an emphasis on interactions between nutrients and between specific psychosocial and nutritional risk factors; the ways in which individual (e.g., gender) or cultural characteristics can moderate nutrition development relations; and a broader range of populations, such as sibling or elderly caregivers, and outcome variables, such as social-emotional development, temperament and mental health.

Kinetic monitoring of enzymatic reactions in real time by quantitative high-performance liquid chromatography-mass spectrometry.

The study of enzyme kinetics under steady-state conditions represents a common and very useful method for investigating the mechanisms of enzymatic reactions. We report the use of mass spectrometry (MS) coupled with HPLC for the kinetic analysis of enzymatic reactions in real time. The hydrolysis of dinucleotides with bovine pancreatic ribonuclease A (RNase A) and the substrate-specific hydrolysis of lactose with beta-galactosidase can be monitored using ion-spray (pneumatically assisted electrospray) mass spectrometry as a sensitive and specific detector for the native substrates. The resulting data can be used to calculate both KM and Vmax for each system. Kinetic parameters obtained for RNase A and beta-galactosidase paralleled those obtained by conventional techniques. These findings suggest the possibility of developing alternative techniques, based on mass spectrometric detection, for performing kinetic analyses of enzymatic processes where no simple spectrophotometric assay is feasible. In addition to enabling the determination of kinetic parameters for authentic substrates, and not chromogenic analogs, such assays would also be useful in situations where very high sensitivity and specificity are desired.

Relation of maternal zinc nutriture to pregnancy outcome and infant development in an Egyptian village.

Zinc nutriture of women living in a periurban Egyptian village was examined over the last 6 mo of pregnancy and the first 6 mo of lactation as one of several potential determinants of pregnancy outcome and infant development. Estimated bioavailable zinc intake was approximately 2 mg/d from diets high in phytate and fiber. Among numerous variables analyzed by multiple regression, early pregnancy weight (3 mo) and plasma zinc concentrations in the second trimester formed the best predictor model of birth weight, accounting for 39% of the variance. Bioavailable zinc intake during pregnancy was part of a profile of micronutrient intakes related to neonatal habituation behavior, a measure of early information processing. Performance on the Bayley motor test at 6 mo of age was negatively related to maternal intakes of plant zinc, phytate, and fiber, suggesting that zinc bioavailability was involved. Maternal dietary intake explained most of the variance observed in infant motor performance; however, predictive variance was amplified by the psychosocial context.

Recent developments in electrospray mass spectrometry including implementation on an ion trap.

Mass spectrometry (MS) may be the ultimate detection technique when combined with modern condensed phase separation sciences. The technique combines sensitivity with excellent specificity, so the pharmaceutical analyst can obtain definitive information regarding components separated in a mixture. Thus, mass spectrometric detection not only provides evidence of a chromatographic peak, but it also provides important information including molecular weight and structural information enabling identification of the components. The coupling of an atmospheric pressure ionization (API) mass spectrometer to most of the separation science techniques offers a simpler alternative from earlier non-routine, less sensitive systems where the vacuum systems struggled to handle the liquid effluent from these systems. Contemporary sensitive and analytically rugged API systems can be operated unattended for extended periods of time thus reducing the cost per sample to a reasonable value especially given the wealth of information provided. Although the mass spectrometer is more complicated than conventional spectroscopic detectors, present day API systems effectively decouple the liquid-phase separation inlet from the high-vacuum system where mass analysis occurs. The ability to form gas-phase ions at atmospheric pressure and sample primarily the analyte ions into the mass spectrometer promises a bright future for combining on-line condensed phase separation science techniques with mass spectrometry. The increasing ease of performing these experiments offers new analytical opportunities for pharmaceutical laboratories.

Relation of rearing environment to adaptive behavior of Egyptian toddlers.

2 questions were explored: (1) Do observed relations found in Western cultures between specific psychosocial environmental factors and toddlers adaptive behavior resemble the pattern of relations found in a non-Western setting? (2) Does the specificity of relations between environment and performance found in Western cultures also operate in a non-Western culture? Subjects were 153 Egyptian toddlers, 18-30 months of age, and their caregivers. Twice a month between 18 and 30 months toddlers were observed in naturalistic interactions with their caregivers, and measures of caregiver behavior and toddler functioning were coded. Replicating previous results from Western cultures, canonical analysis indicated that caregiver vocal stimulation was positively related to indices of toddler behavioral competence, while nonverbal response to vocalization and physical contact stimulation were negatively related. The salience of sib caregivers was also noted. Particularly for the age period between 24 and 29 months, results indicated specificity of environmental action such that measures of caregiver vocal stimulation were uniquely related to measures of toddler vocalization, while caregiver response to distress was uniquely related to toddler emotionality. The present pattern of results suggests at least some degree of cross-cultural generalizability of environment-development relations and of the specificity model of environmental action.

Relations between nutrition and cognitive performance in Egyptian toddlers.

PIP: Two relatively unexplored questions--which specific components of chronic mild child malnutrition are associated with specific behavioral and cognitive deficits and do psychosocial child rearing factors moderate the nutrition-development relationship?--were investigated in a longitudinal study involving 153 toddlers from the Egyptian village of Kalama. Beginning at study entry at age 18 months and continuing until 30 months, food intake information was obtained for 2 consecutive days each month. During the same period, researchers visited the homes twice a month to observe both toddler behavior and caregiver styles. Finally, standardized infant development tests were administered at 24 months and 30 minutes and the capacity for exploratory, symbolic play was assessed. Specific components of nutritional intake at 18-23 months of age were confirmed to be uniquely related to specific developmental outcomes at 24 months of age. Changes in toddler intake from 18-30 months also predicted 30-month cognitive performance. At 24 months, general mental competence was associated positively with fat intake, total kilocalories, and total protein intake while symbolic play capacity was related only to total protein intake. Significant increases in predictive variance occurred when the model was expanded to encompass caregiver behaviors as well as nutritional factors. For example, when nonverbal response to child vocalizations was used as the environmental measure, there was evidence of both coactive and synergistic interactions for comparisons involving cognitive development indicators. For symbolic play, an increase in predictive variance was found when the model included the multiplicative interaction of animal kilocalories by nonverbal response to vocalizations. Also observed was a buffering effect against poor psychosocial rearing conditions given the presence of adequate nutritional intake. These findings suggest the feasibility of multidimensional risk profiles, the provision of specific nutrients targeted at specific deficits, and multidisciplinary interventions.^ieng

Diet during lactation associated with infant behavior and caregiver-infant interaction in a semirural Egyptian village.

Potential processes through which nutritional and non-nutritional factors can relate to infant state and behavior and mother-infant interactions were examined in 41 mother-infant pairs from semirural Egyptian households. All infants were breast-fed, and breast milk was the main source of their nutrient intake. Median birth weight was close to reference median; however, most infants showed growth faltering when they were 3-6 mo of age. Among the infant behavioral and state variables, only drowsiness (a proxy for activity and alertness) was significantly associated with the nutritional and non-nutritional factors examined. Among these factors, mothers' intakes of animal source foods and certain B vitamins were the strongest predictors of drowsiness. The nature of the association between maternal diet and drowsiness, examined by multiple regression analysis, showed clearly that inadequate diet of the mother was the major risk factor. Alertness of infants was further compromised when there were several children in the households. The small, less vocal and less alert infants received less vocalization from their mothers. In this environment, infants of undernourished mothers may not receive the extra care and stimulation needed and are at risk for subsequent developmental disabilities.

PIP: Each week physicians visited the households of 41 mother-infant pairs living in Kalama, Egypt, to examine infant behavior and caregiver-infant interactions from 3 to 6 months of age, as they relate to various factors, e.g., maternal nutrition and health and socioeconomic status. All the mothers breast fed their infants. Liquids which are inferior sources of energy and nutrients were the most customary food supplements during the first 6 months. Even though the median birth weight tended to equal that of the WHO international reference, most infants experienced faltering growth by 3 months. At 6 months, 75% were below the 25th percentile and 20% were in the 5th percentile. Growth faltering was linked with increased diarrhea (p .05). Even though the mothers' diets contained sufficient niacin, thiamin, folate, and vitamin C, the diets did not supply mothers enough riboflavin, vitamin B-6, vitamin A, calcium, and zinc. Breast milk did not have adequate amounts of vitamin B-6 and, perhaps, not even enough riboflavin and vitamin A. It did contain adequate amounts of calcium, however. Drowsiness was significantly related to maternal diet during lactation but not during pregnancy (especially energy intake from animal sources, p = .0001; energy intake from plant sources, p = .03); number of siblings (p = .009); crowding (p = .06); vocalization from mothers (p = .08); and low socioeconomic status (p = .07). Maternal diet was the best predictor of drowsiness followed by number of siblings. Without remediation in maternal nutrition, infants of undernourished mothers appeared not to receive appropriate care and stimulation, therefore placing them at risk of subsequent development disabilities. Intervention studies should be done to examine this main effect of maternal undernutrition and the importance of covarying environmental risk factors, e.g., crowding.

Infants with myelomeningocele: visual recognition memory and sensorimotor abilities.

The cognitive development was explored of 29 infants with myelomeningocele. Although as a group they tended to be less likely to demonstrate visual recognition memory than a matched control group, excluding the more severely cognitively impaired infants from analysis rendered this difference non-significant. Infants with myelomeningocele demonstrated more verbal imitation on the Uzgiris-Hunt Assessment Scales than the controls, but there were no statistically significant differences between the groups on the Objects Permanence, Schemes, or Objects in Space subscales. The two groups did not differ on the majority of demographic and environmental variables examined, suggesting that group differences may be due to differences in biomedical variables. However, within the myelomeningocele group, environmental variables correlated with the majority of the cognitive measures, while correlations between cognitive performance and both demographic and biomedical variables were at chance level.

Determinants of pregnancy outcome and newborn behavior of a semirural Egyptian population.

Several potential determinants of birth weight and neonatal behavioral organization, ie, maternal anthropometry, food intake (energy, protein, and plant- and animal-source foods), morbidity, and household socioeconomic status, were followed systematically in a semirural Egyptian population during greater than or equal to 6 mo of pregnancy. In early pregnancy mothers were generally normal weight to moderately overweight. Their mean energy intake, largely from plant sources, was approximately 8.37 MJ/d (2000 kcal/d) during trimesters 2 and 3. Early (3 mo) pregnancy weight and weight gain during trimesters 2 and 3 were significantly positively related to birth weight Z scores. The best predictor model examined for birth weight included early pregnancy weight, weight gain, and length of gestation (R2 = 0.45). Early pregnancy weight and maternal intake of animal-source foods were significant positive predictors of the newborn's orientation and habituation behavior, respectively. Habituation and orientation measures assess the infant's early ability to process information.

Vitamin B-6 status of Egyptian mothers: relation to infant behavior and maternal-infant interactions.

Functional consequences of marginal maternal vitamin B-6 status for behavior of the neonate and for mother-infant interactions at age 3-6 mo were assessed by a double-blind procedure. In 27 of 70 Egyptian village women studied, vitamin B-6 concentration of their milk was considered indicative of poor maternal vitamin B-6 nutriture. Neonatal behavior, quantified by the Brazelton Neonatal Behavioral Assessment Scale, showed that consolability, appropriate build-up to a crying state, and response to aversive stimuli were significantly correlated with maternal vitamin B-6 nutriture. Naturalistic observational procedures, used twice monthly with infants aged 3-6 mo, indicated that mothers assessed as having marginal vitamin B-6 status were less responsive to their infants' vocalizations, showed less effective intervention to infant distress, and were more likely to use older siblings as care-givers than were mothers of better vitamin status. We conclude that vitamin B-6 was a factor influencing both the behavior of the mother and her infant.

Urokinase administration in pediatric patients with occluded central venous catheters.

The effectiveness of urokinase in declotting central venous catheters was retrospectively assessed in a pediatric population of 63 patients ranging in age from 4 days to 22 years (mean, 6.4 years). Results of treatment from 103 episodes of catheter occlusion were evaluated. Urokinase was instilled into occluded single-lumen catheters and into each lumen of double-lumen catheters, as well as into occluded implanted ports. Patency was restored to 101 of 103 occluded catheters, with no side effects observed subsequent to clearance of the catheters with the urokinase infusion. Significant cost savings were seen using urokinase to restore patency as compared to the cost of replacing the catheter. These data clearly support the use of urokinase in a pediatric population as a safe and cost-effective alternative to catheter replacement in cases of thrombotic occlusion.

Infant temperament and subject loss in a habituation procedure.

It has been assumed that subject loss in habituation procedures is most likely due to random factors such as the behavioral state of the infant during testing. This study explored the possibility that infant temperament might contribute to subject loss. 114 infants at 3 age levels (11, 18, and 28 weeks) were rated by their mothers on a temperament questionnaire and then subjected to a habituation procedure. Analyses showed that female infants who were unable to complete the habituation task were reported as being more fussy and unadaptable. Female infants were less likely to complete at least part of the procedure than were male infants. The results suggest that subject loss in habituation studies may be the result of nonrandom individual difference factors and not just the result of temporary fluctuations in state.

The use and abuse of environment in behavior-genetic research.

The measurements and conceptualizations of environment by environmentally oriented behavioral scientists and behavior geneticists are contrasted. For the environmentalist, the measurement of environment involves direct observation are manipulation. The environment is conceptualized as multidimensional, dynamic and transactional in nature, as well as being mediated, in its effects, by the individual. In contrast, in behavioral-genetic studies the environment is either estimated but not measured or is only measured indirectly. The conceptualization of environment used by behavior geneticists is static and global. In spite of these differing conceptualizations and measurements, areas of common interest are observed, centering around the question of how genotype and environment transact to influence development. Specifically, commonalities and joint research strategies are proposed for the areas of gene-environment correlation/transactional model and gene-environment interaction/organismic specificity.