Profiling of impurities in illicit methamphetamine by high-performance liquid chromatography and capillary electrochromatography.

High performance liquid chromatography (HPLC) with photodiode array (PDA) UV and fluorescence (FL) detection, and capillary electrochromatography (CEC) with laser-induced fluorescence (LIF) detection were investigated for the analysis of acidic extracts derived from illicit methamphetamine. These compounds include major impurities from the hydriodic acid/red phosphorous reduction method, i.e., 1,3-dimethyl-2-phenylnaphthalene and 1-benzyl-3-methylnaphthalene, and other trace-level, structurally related impurities. For certain of these solutes, HPLC with conventional FL detection gave at least a 60x increase in sensitivity over UV detection. In addition, other highly fluorescent impurities were detected in methamphetamine produced via four other synthetic routes. The use of a rapid scanning FL detector (with acquisition of "on the fly" excitation or emission) provided structural information and gave "optimum" excitation and emission detection wavelengths. CEC with LIF detection using UV laser excitation provided greatly improved chromatography over HPLC, with good detection limits in the low ng/ml range. Both methodologies provide good run-to-run repeatability, and have the capability to distinguish between samples.

Single-system ureteroceles in infants and children: imaging features.

PURPOSE: The purpose of this manuscript is to describe the clinical and imaging findings in children who have single-system ureteroceles. MATERIALS AND METHODS: We reviewed the urology records and imaging studies in 32 consecutive infants and children who were diagnosed in our department with single-system ureteroceles. RESULTS: There were 35 ureteroceles in the 32 patients-- 29 were unilateral (14 right-sided, 15 left-sided) and 3 were bilateral. Twenty-five patients were boys (78 %) and 7 girls. Mean age at presentation was 0.7 years (0-9.2 years). Prenatally detected hydronephrosis or cystic renal dysplasia was the most common presentation (24 patients). Four presented with urinary infection, 2 with abdominal mass, 1 had myelomeningocele, and 1 had hypospadias. Three patients also had multiple non-urologic, congenital anomalies. Thirty-three ureteroceles were intravesical, and 2 were ectopic to the bladder neck. Twenty-four ureteroceles were associated with ipsilateral hydroureteronephrosis and 10 with ipsilateral multicystic dysplastic kidney. One patient had a normal ipsilateral kidney and a contralateral multicystic dysplastic kidney. The ureterocele was identified on at least one imaging study in each patient. Sixteen ureteroceles (47%) everted at VCUG, mimicking paraureteral diverticula. Other variations included ureterocele prolapse and inadvertent ureterocele catheterization (1 each). CONCLUSIONS: Single-system ureterocele is an important, although uncommon cause of hydronephrosis and renal dysplasia in infants and children. Single-system ureterocele is distinguished clinically from the more common duplex-system ureterocele by its frequent occurrence in boys and its association with multicystic dysplastic kidney. Because these ureteroceles are frequently small and have a propensity to evert at VCUG, they can be mistaken for paraureteral diverticula.

Alteration of computer dental radiography images.

This study was designed to determine if digital images stored on the hard drive of a Schick computer dental radiography system could be exported, altered, and then restored to the drive without any visible signs of alteration. Digital images were downloaded from the computer dental radiography system using an I-Omega Zip Drive, 100-MB capacity, and then opened in Corel Photo Paint where images were altered and manufacturer export symbols were edited. The resulting images were printed to a default printer (Fargo Foto Fun). The ease of manipulation of the exported digital images reflects the need for the manufacturer to implement safeguards so that the integrity of digital imaging cannot be compromised. Computer dental radiography has many advantages: conservation of time (instant radiographs), less radiation (50 to 60%), no chemical waste, and many viewing options. However, questions that might be raised regarding the ability of persons with minimal computer expertise, using a commercially available program to alter images should be addressed.

Characterization of replication-competent adenovirus isolates from large-scale production of a recombinant adenoviral vector.

Replication-deficient adenoviral vectors have been developed for the delivery of DNA sequences encoding a variety of proteins intended for the management of disease through gene therapy. One concern is the occurrence of replication-competent adenovirus (RCA) in the population of replication-deficient adenoviral vectors as a result of recombination or contamination. To address this concern, it is necessary to determine the frequency of occurrence and to fully characterize the molecular structure and biological infectivity of RCA. rAd/p53 is a pIX-deleted p53 gene therapy vector that is designed to lower the RCA occurrence and to deliver the tumor suppressor gene p53 for treatment of various cancers. Multiple preparations of the replication-deficient adenoviral vector rAd/p53 were tested for the presence of RCA, employing a sensitive biological assay. Single plaques from RCA-positive preparations of rAd/p53 were isolated for molecular characterization. All of the RCA isolates displayed a single unique structure that contains the complete E1 sequence of adenovirus type 5 but lacks the p53 sequence. The detailed sequence analysis of the RCA suggests that it is most likely generated as a result of recombination events between the rAd/p53 DNA and the 293 host adenoviral sequence. Results from viral infectivity analysis by flow cytometry demonstrate no substantial difference in infectivity of RCA, rAd/p53, and wild-type adenovirus type 5 in 293 cells.

Effects of various anionic chiral selectors on the capillary electrophoresis separation of chiral phenethylamines and achiral neutral impurities present in illicit methamphetamine.

In this study, various anionic chiral selectors were investigated for the capillary electrophoresis (CE) separation of six chiral phenethylamines and three achiral neutral impurities which are commonly identified in illicit methamphetamine. Analyses were carried out at pH 8 (high osmotic flow) with untreated capillaries using 25 mM chiral surfactant or 10 mM charged cyclodextrin. The chiral selectors included the micelle (R)-N-dodecoxycarbonylvaline (EnantioSelect (R)-Val-1) (ES) and the cyclodextrins sulfobutyl(IV)-ether-beta-cyclodextrin (SBE(IV)-beta-CD) (BSB4), SBE(VII)-beta-CD (BSB7), SBE(XII)-beta-CD (BSB 12), SBE(IV)-gamma-CD (GSB-4), SBE(VII)-gamma-CD (GSB-7), sulfated(XI)-alpha-cyclodextrin (SU(XI)-alpha-CD (AS11), SU(VII)-beta-CD (BS7), SU(XII)-beta-CD (BS12) and SU(XIII)-beta-CD (GS13). Enantiomeric and achiral selectivity strongly depends on the size of the CD, the average degree of substitution, and the type of substitution. ES exhibits good performance for the neutral solutes, but exhibits enantiomeric selectivity only for the alpha-hydroxyphenethylamines. GS13 provides the best overall enantiomeric selectivity. All fifteen solutes related to methamphetamine are simultaneously separated using BSB7.

Capillary electrophoresis analysis of isomeric truxillines and other high molecular weight impurities in illicit cocaine.

The analysis of by-products and impurities in illicit cocaine, including the isomeric truxillines, is important for derivation of both strategic and tactical intelligence. In the present study, various capillary electrophoresis techniques were investigated for this purpose. The use of the anionic beta-cyclodextrin sulfobutyl ether IV as a run buffer additive at pH 8.6 gave a good separation of the truxillines and similar high molecular weight impurities in less than eight minutes. These impurities were first isolated from the bulk cocaine matrix using liquid-liquid extraction and size-exclusion high performance liquid chromatography. There was a red shift in the UV spectra obtained for the truxillines using photodiode array (PDA) UV detection during CE analysis. This anomalous behavior is attributed to photo-degradation of the truxillines during the PDA-UV irradiation process. Laser-induced fluorescence detection using a UV krypton/fluoride laser provided greater selectivity and sensitivity versus UV detection for certain uncharacterized high molecular weight impurities.

Cocaine Profiling Methodology - Recent Advancesk.

The rationale for developing cocaine profiling methodology is described. Current cocaine signature procedures in use at the U.S. Drug Enforcement Administration's Special Testing and Research Laboratory are reviewed. Newer selective and sensitive methodology, recently developed, is described. That methodology detects more alkaloidal impurities in refined illicit cocaine than heretofore reported. The alkaloidal impurities were isolated from the bulk cocaine matrix by alumina column chromatography and detected using capillary gas chromatography-mass selective detection in the selected ion mode. Fifty-one refined illicit cocaine samples were subjected to this methodology for the determination of 15 selected alkaloids. Reproducibility data are reported. Methodology for the isolation, detection, and characterization of coca alkaloids in South American coca leaf, a commercial coca-leaf extract, and a large seizure of refined illicit cocaine is reviewed.

Novel chlorinated tropanes derived from the treatment of cocaine with sodium hypochlorite.

Several novel chlorinated tropanes were produced when cocaine was treated with aqueous sodium hypochlorite. Two of these, 2'- and 3'-chlorobenzoyloxy-2-carbomethoxypseudotropine (that is, ortho- and meta-chlorococaine), were characterized by synthesis and gas chromatography/mass spectrometry. Four other new chlorinated tropanes (endo-6- and 7-chlorococaine, exo-6- or 7-chlorococaine and N-chlorobenzoylnorecgonine methyl ester) were also tentatively identified via their gas chromatography retention data and mass spectra. The results are of potential use in cocaine signature and comparative analysis.

Separation and detection of acidic and neutral impurities in illicit heroin via capillary electrophoresis.

The separation and detection of acidic and neutral impurities in illicit heroin using capillary electrophoresis (CE) is described. Separations were achieved using charged cyclodextrin modified micellar electrokinetic capillary chromatography. The use of the anionic beta-cyclodextrin sulfobutyl ether 1V in combination with sodium dodecyl sulfate significantly increased resolution. Improved selectivity and/or sensitivity in detection was obtained using photodiode array ultraviolet and laser-induced fluorescence detection. The phenanthrene-like heroin impurities exhibit high native fluorescence under krypton-fluoride laser excitation (248 nm). The limit of detection by laser-induced fluorescence detection for one of these solutes (acetylthebaol) is 1.8 ng/ml, 500 times more sensitive than UV. This methodology is applicable to analysis of both crude and refined heroin.

Detection and determination of pseudococaine in coca leaves and illicit cocaine samples.

Methodology is presented for the isolation, identification and determination of pseudococaine in coca leaves and illicit cocaine. Coca leaves, crude cocaine base (coca paste), refined cocaine base and refined cocaine hydrochloride, all derived from the same geographic location in Bolivia, were examined. Pseudococaine and other coca alkaloids were isolated from leaf samples using toluene extraction followed by acid/Celite trap and ion-pair column chromatography, and from crude and refined cocaine samples by acid/Celite column ion-pairing chromatography. Mass spectral analysis of coca leaf isolates confirmed the presence of pseudococaine. Pseudococaine was quantified by capillary gas chromatography with flame ionization detection at levels of 0.0001-0.035% (relative to cocaine) in refined illicit cocaine and coca leaves.

In-depth chromatographic analyses of illicit cocaine and its precursor, coca leaves.

Chromatographic methodology used for the in-depth alkaloid analyses of coca leaves and for the characterization of alkaloidal impurities and manufacturing by-products in illicit refined cocaine samples is reviewed. This includes liquid-liquid partition and liquid-solid adsorption column chromatography, packed- and capillary-column gas chromatography with flame-ionization, electron-capture, nitrogen-phosphorous and mass spectrometric detection, and high-performance liquid chromatography with ultraviolet detection. The rationale supporting the presence and determination of processing impurities/by-products in cocaine samples is discussed, and chromatographic methodology used for the development of drug impurity signature profiles is presented.

Determination and in-depth chromatographic analyses of alkaloids in South American and greenhouse-cultivated coca leaves.

Methodology is described for the detection and/or determination of cocaine and minor alkaloids in South American coca as well as in greenhouse- and tropical-cultivated field coca of known taxonomy. Coca leaf from Bolivia, Peru, Ecuador and Colombia were subjected to the determination of cocaine, cis- and trans-cinnamoylcocaine, tropacocaine, hygrine, cuscohygrine and the isomeric truxillines. The greenhouse samples were cocaine-bearing leaves of the genus Erythroxylum and included E. coca var. coca, E. novogranatense var. novogranatense and E. novogranatense var. truxillense, and the alkaloids determined were cocaine, ecgonine methyl ester, cuscohygrine, tropacocaine and the cinnamoylcocaines. The tropical-cultivated coca were E. novogranatense var. novogranatense and E. coca var. coca. Cocaine and minor alkaloids were isolated from basified powdered leaf samples using a toluene extractant, followed by acid-Celite column chromatography. The isolated alkaloids were determined by capillary gas chromatography with flame ionization or electron-capture detection. Methodology is also presented for the isolation and mass spectral analysis of numerous trace-level coca alkaloids of unknown structure.

A computerized neural network method for pattern recognition of cocaine signatures.

This article describes a practical procedure for rapidly searching a large database of cocaine signatures to identify database entries that closely resemble a given reference cocaine exhibit using a personal computer (PC). The procedure takes advantage of the pattern recognition capability of the multilayer perceptron neural network to identify similar cocaine signatures. A PC-based software implementation is now being used on a daily basis at the North Carolina State Bureau of Investigation (NCSBI) to aid forensic experts in identifying signatures that originate from the same batch. Intelligence reports generated from database searches have been useful to undercover agents in the field who are striving to build drug related conspiracy cases. This software was developed as a collaborative effort between the NCSBI and the Center for Systems and Engineering of the Research Triangle Institute.

Illicit Production of Cocaine.

The predominant methods currently used for illicit production of cocaine are described. For illicit natural cocaine (i.e., from coca leaf), this includes production of coca paste from coca leaf via both the solvent and acid extraction techniques, purification of coca paste to cocaine base, and conversion of cocaine base to cocaine hydrochloride. For illicit synthetic cocaine (i.e., synthesized from precursor chemicals), the classic five-step synthetic route used in all clandestine laboratories seized to date is summarized. The origins of the most common alkaloidal impurities and processing/synthetic by-products typically identified in illicit natural, illicit synthetic, and pharmaceutical cocaine are discussed. Forensic differentiation of exhibits arising from the various production methods are addressed both in terms of overall product purity and the presence/absence of these impurities and by-products.

Modification of an automated vascular diagnostic system for hyperbaric use.

Modifications of an automated, noninvasive vascular diagnostic system (VASCULAB, MedaSonics, Inc.) for measuring blood pressure and plethysmographic blood flow responses to normobaric and hyperbaric oxygenation are described. The system consisted of a pump for inflating and deflating blood pressure cuffs and a microprocessor program controller (VSC21) with ultrasound Doppler, strain-gauge plethysmograph, and chart recorder. Inclusion of the VSC21 controller in the chamber was required for performance of procedures that could not be controlled from outside the chamber. All other components were outside the chamber. For fire prevention the VSC21 controller was nitrogen-purged in an acrylic case mounted on a mobile cart. Pressure-cuff tubes were attached via adapted fittings and connectors in the cart to connector ports in the controller's front panel. Electrical power cables and instrument signal wires were routed through chamber penetrations to an electrical power source and other VASCULAB components, respectively, outside the chamber. Initially, compression of the chamber to pressures in excess of 1.68 bar disabled the VSC21, requiring removal of its front membrane panel and ventilation of its pressure-sensitive keypad switches. This allowed automated assessment of blood pressure and calf blood flow at test pressures of 1.97 and 2.96 bar.

Neutrophil cathepsin G increases permeability of cultured type II pneumocytes.

Neutrophils contribute to inflammatory injury in some types of acute and chronic lung disease. Among the injurious agents released by activated neutrophils are cationic proteins such as cathepsin G that may contribute to edema formation. Using cultured rat type II alveolar epithelium, we examined the effect of human neutrophil cathepsin G on permeability to mannitol and electrical resistance across the cultured epithelium. Monolayers maintained in control media alone had constant permeability to mannitol (4.5 +/- 0.3 X 10(-3) cm/h at base line and 4.4 +/- 0.3 X 10(-3) cm/h after 2 h). When monolayers were exposed to cathepsin G at concentrations of 5, 10, and 20 micrograms/ml, there was a progressive increase in mannitol permeability (120 +/- 26% increase at 5 micrograms/ml, 174 +/- 40% increase at 10 micrograms/ml and 301 + 116% increase at 20 micrograms/ml, P less than 0.001). The effect of cathepsin G on mannitol permeability was blocked by the presence of the polyanion-sulfated dextran, and the polycation protamine also increased permeability to mannitol 57 +/- 5% (P less than 0.001), suggesting a role for charge in this effect. Cathepsin G exposure also resulted in a progressive decrease in electrical resistance (67 +/- 2% base line after 15 min, 55 +/- 1% after 30 min, and 43 +/- 2% after 60 min). Cathepsin G at these concentrations was not cytolytic to the cells as measured by lactate dedrogenase release. These functional alterations were accompanied by increase in intercellular gaps in the monolayers seen on scanning electron microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of type II alveolar epithelial cells by flow cytometry and fluorescent markers.

Type II alveolar epithelial cells play a crucial role in maintaining the structure and functions of pulmonary alveoli. A number of techniques have been described to isolate type II cells for in vitro studies; however, type II cell suspensions isolated by each technique are still contaminated by macrophages or monocytes. The present studies describe the use of flow cytometry to accurately characterize the composition of these cell suspensions. With freshly isolated type II cell suspensions, type II cells could be distinguished from macrophages and monocytes by two methods: (1) the combination of natural fluorescence and orthogonal light scatter, or (2) the use of monoclonal antibodies OX-1 (directed against a common leukocyte antigen present on rat macrophages and monocytes) and PKK-1 (directed against cytokeratin intermediate filaments present in type II cells). With cultured type II cells, the combination of natural fluorescence and orthogonal light scatter did not distinguish between type II cells and macrophages or monocytes; however, the monoclonal antibodies OX-I and PKK-1 continued to distinguish between these cell types. As an example of the use of these techniques, the methods were used to define the sequential expression of class I and II major histocompatibility antigens on both type II cells and on macrophages or monocytes in the same cell preparations. These methods are of potential value in isolating pure populations either of type II cells or of macrophages or monocytes by cell sorting and in accurately identifying the cells present in type II cell suspensions or cultures.

Neutrophil adherence to human endothelial cells.

These studies evaluated whether the increased adherence of neutrophils to endothelium after exposure to lipopolysaccharide (LPS) endotoxin is primarily an effect on neutrophils or on endothelial cells. The studies demonstrate that preincubation of monolayers of human umbilical vein endothelial cells with LPS has a significantly greater effect on neutrophil adherence to endothelium than does preincubation of neutrophils with LPS (P less than 0.001 for each amount of LPS). Although the effect was small compared with incubation of endothelial cells with LPS, incubation of neutrophils with LPS did significantly increase their subsequent adherence to endothelial cells compared with controls (P less than 0.05). LPS was not toxic to either endothelial cells or neutrophils, as measured by the release of lactate dehydrogenase. Preincubation of endothelial cells with LPS at a concentration of 1.0 to 10 micrograms/ml maximally increased their ability to bind to neutrophils, and this effect was maximally expressed after 4 hours of exposure to LPS. In the assay, neutrophil binding to LPS-stimulated endothelial cells was rapid and did not increase after 30 minutes of coculture of neutrophils and endothelium. Morphologic studies demonstrated that LPS opened cell-to-cell junctions between endothelial cells. Neutrophils that attached to these monolayers of LPS-stimulated endothelial cells bound, primarily, to the margins of the endothelial cells and not to the underlying tissue culture dishes, which were exposed after incubation with LPS. These observations suggest that LPS increases neutrophil adherence primarily, but not solely, via an effect on endothelial cells.