Internal Reference Standard Research Articles

Stability - Indicating Method For The Determination of Bromazepam And Delorazepam Via Proton Magnetic Resonance Spectroscopy

A new, simple proton magnetic resonance method (1HNMR) for the rapid and direct quantitation of intact bromazepam and delorazepm in presence of their acid - induced degradation products is presented. DMSO - d6 is used as a solvent and maleic acid as internal reference standard. The resonance peak of the two hydrogens of the methylene group of each drug near 4.2 ppm is integrated as a measure for the drug in comparison to the sharp singlet of maleic acid at about 6.25 ppm. The proposed procedure gives accurate and reproducibe results when applied for the anlaysis of both drugs either per se, in mixtures with their degradation products or in their tablets.

Flow cytometric analysis of nuclear genome of the Ethiopian cereal tef [Eragrostis tef (Zucc.) Trotter

Flow cytometric analysis of nuclear DNA content was performed by using nuclei isolated from young leaf tissue of tef (Eragrostis tef). The method was very useful for rapid screening of ploidy levels in cultivars and lines of tef representing the phenotypic variability of this species in Ethiopia. The results of the analysis showed that all cultivars were tetraploid. Flow cytometry was also used to determine nuclear DNA content in absolute units (genome size) in four tef cultivars. Nuclei isolated from tomato (Lycopersicon esculentum, 2C=1.96 pg) were used as an internal reference standard. The 2C DNA content of individual tef cultivars ranged from 1.48 to 1.52 pg (1C genome size: 714 Mbp-733 Mbp), the differences among them being statistically nonsignificant. The fact that the nuclear genome of tef is only about 50% larger than that of rice should make it amenable for analysis and mapping at the molecular level.

Proton nuclear magnetic resonance determination of hexamethylenetetramine in the presence of formaldehyde and urine

A new proton NMR method has been developed for micro-amount determination of aqueous hexamethylenetetramine (HMT) alone, in the presence of large amounts of formaldehyde, and in urine. The method is based on the direct determination of HMT in [2H3]acetonitrile containing anisole as the internal reference standard. HMT is determined in the range 5–50 µg using a 5 mm NMR tube.

DNA levels in somatic and germ line cells of a freshwater crustacean, Mesocyclops edax

Chromatin diminution--the fragmentation and elimination of chromosome Regions--provides an unusual opportunity to study genomic reorganization during development. Some species of copepods regularly excise and discard large amounts of nuclear DNA from presumptive somatic cell lines during early cleavage stages . To study this phenomenon in M. edax we determined DNA-Feulgen levels for more than 5,600 individual nuclei from squash preparations of 30 female and 25 male adults collected from lakes in Nova Scotia, Virginia and Florida. Fixation in 3:1 methanol/acetic acid was followed by squashing individual specimens in 45% acetic acid, freezing each slide in liquid N2 and thawing in absolute ethanol before air drying. Each series of slides was stained with the feulgen reaction for DNA and measured with a Vickers M86 scanning and integrating microdensitometer at 560 nm, using chicken RBC nuclei as an internal reference standard of 2.5 pg DNA per cell. This allowed us to ask several questions: are there differences in genome size (1) among specimens from different collecting localities, (2) between females and males at any single locality, and (3) between cells of germ line and somatic cell lineages?

Refinement of unit-cell parameters by whole-powder-pattern fitting technique

The accuracy of the unit-cell parameters refined by using the whole-powder-pattern decomposition method is discussed. Powders of W, ZnO, TiO2, BaTiO3 Mg2SiO4, Al2SiO5 (+α-SiO2), and monoclinic ZrO2 were used as test samples. Two internal standard reference materials of Si and CeO2 and two types of powder diffractometers were used for data collections. The systematic peak-shift was corrected by determining the unit-cell parameters and the error function simultaneously during the whole-pattern-fitting. The estimated standard deviations for sample means ranged from <10 ppm (10−6) in cubic symmetry to 20∼50 ppm in monoclinic symmetry. These analyses could be carried out almost automatically in a computation time of less than l min for each sample on a workstation. The use of symmetric experimental profiles, obtained by the suppression of axial divergence, is very effective and of essential importance for improving the accuracy of unit-cell parameters.

Pulmonary regurgitation in the late postoperative follow-up of tetralogy of Fallot. Volumetric quantitation by nuclear magnetic resonance velocity mapping.

Pulmonary regurgitation frequently occurs after surgical correction of tetralogy of Fallot. To date, reliable quantitation of pulmonary regurgitation has not been possible, and therefore the clinical significance of pulmonary regurgitation is controversial. Nuclear magnetic resonance (NMR) velocity mapping allows accurate measurement of volumetric flow. The feasibility and accuracy of NMR velocity mapping to quantify pulmonary regurgitation volumes are studied in patients after Fallot repair. In 18 patients (mean age, 16.5 +/- 6.5 years), late (12.6 +/- 5.2 years) after Fallot surgery, forward and regurgitant volume flow was measured in the main pulmonary artery with NMR velocity mapping. To validate the measurements of pulmonary forward flow, right ventricular stroke volume was used as an internal reference standard. Pulmonary regurgitation volumes were compared with the differences between the corresponding right and left ventricular stroke volumes. Ventricular volumes were measured with a multisection gradient echo NMR method. In addition, the relation between pulmonary regurgitation and right ventricular volumes was studied. Measurements of pulmonary regurgitation volume with NMR velocity mapping closely corresponded with the tomographically determined volumes (r = .93). Forward pulmonary volume flow was nearly identical to right ventricular stroke volume (r = .98). Pulmonary regurgitation volume was significantly correlated with end-diastolic volume (r = .82, P < .0005), end-systolic volume (r = .63, P < .01), and stroke volume (r = .89, P < .0005) of the right ventricular but not with right ventricular ejection fraction (r = .41, P = NS). NMR velocity mapping is an accurate method for the noninvasive, volumetric quantification of pulmonary regurgitation after surgical correction of tetralogy of Fallot.

Stability of propidium iodide stained samples by the coulterR DNA prep workstation for analysis by flow cytometry

At the Caulfield General Medical Centre, paraffin-embedded specimens analysed for DNA ploidy are prepared by a modification of the Hedley Technique'. Nuclear preparations are stained by the COULTER^R DNA Prep Reagent System. This is a commercial workstation designed to prepare cell or nuclear suspensions for quantitative determination of nuclear DNA content by Flow Cytometry (FCM). The reagents comprise non-ionic detergents for cell lysing and permeabilisation, Propidium Iodide (PI) for DNA staining, and avian erythrocyte nuclei as an internal reference standard. The manufacturers instructions specify that to obtain optimal coefficients of variation (CV's), samples should be incubated in PI at room temperature (RT) for 15 - 17 minutes prior to analysing by FCM and must bel analysed within 2 hours of staining. This investigation set out to determine the stability of the stained specimens beyond this 2 hour limit. After routine analysis of a group of stained samples, the batch was re-analysed at different intervals ranging from 24 hours to 14 days. Samples were stored at RT and protected from light. The values from the histograms collected and compared over this period included: the DNA Index (Dl) : percent half peak CV's (%HPCV); mean channel fluorescence of the major peak(s) in the histogram; and percent of events found in each peak(s). Examination of the histograms after 24 hours demonstrated no alteration in the Dl of the specimen, an average reduction of 10% in the %HPCV, 10% increase in the mean channel fluorescence and a 5% decrease in the percent of events found in the peaks. Re-analysis over a two week period showed these values to remain stable. Less than 10% of the samples deteriorated and were uninterpretable at the end of 14 days. The data shows that stained nuclear preparations are stable beyond the time recommended by the manufacturers and allows for storage of stained specimens with minimal deterioration.

Genome Size in Beaufort Sea Coastal Assemblages of Arctic Ciscoes

Intraspecific variation in nuclear DNA content (genome size) was measured for the Arctic cisco Coregonus autumnalis by fluorescence flow cytometry with the fluorochrome 4′,6-diamidino-2-phenylindole (DAPI). Frozen erythrocytes from the domestic chicken Gallus domesticus were analyzed simultaneously with Arctic cisco samples, providing an internal reference standard. Mean DNA content for 140 Arctic ciscoes from seven sites along the Beaufort Sea coast of Alaska and Canada was 5.99 pg/cell. Sample variances and mean DNA content were significantly heterogeneous among coastal sites. A significant increasing linear trend in mean DNA content was found on a west-to-east cline along the Beaufort Sea coast. Intraspecific variation in genome size observed in this study was substantial, which indicates that genome size characterizations of species based on small samples from a single population may be grossly inadequate.

Accurate Determination of Unit-Cell Parameters using Conventional X-Ray Powder Diffractometry

AbstractA procedure for the accurate determination of unit-cell parameters using conventional Xray powder diffractometry is described. Two important factors in the procedure are: 1) the use of high-resolution-type diffractometer, which can suppress the axial beam divergence and thus gives nearly symmetric diffraction profiles in the low 2θ region and 2) the use of a new algorithm for systematic peak shift correction during the least-squares determination of unit-cell parameters of a sample with an internal standard [Toraya &amp; Kitamura (1990). J. Appl. Cryst. 23 , 282-285]. The procedure has been tested by measuring successively the unit-cell parameter of W, CeO2, and Si in three mixtures, Si+W, W+CeO2, and CeO2+Si: the unit-cell parameter of W, which was first determined by using NIST SRM 640b Si powder as an internal standard reference material, was used as a standard reference value to determine the unit-cell parameter of CeO; in the next W+CeO2 mixture, and so on. The difference between the end value of observed Si unit-cell parameters and the starting value of 5.430940(35) Å were just 1 to 5 p.p.m. High accuracy is attainable in measuring the uni-cell parameters even with the conventional powder diffractometry provided with the nearly symmetric diffraction profile and the algorithm for peak shift correction used in the present study.

Use of Carbon-13 Labelled Dioxins Mixture for Analysis of Polychlorinated Dibenzo-p-Dioxins in Environmental Samples by GC-MS

Abstract Positive identification and quantitation of polychlorinated dibenzo-p-dioxins (PCDD) in complicated environmental samples is described using a C-13 labelled dioxin mixture as an internal reference standard. Environmental samples are spiked with the C-13 labelled dioxin mixture and monitored for labelled and unlabelled dioxins using GC-MS in the electron impact selected ion monitoring (EISIM) mode. The C-13 labelled dioxin mixture and a municipal solid waste (MSW) incinerator fly ash extract show the same number of isomers in each tetra to octa-chlorodioxin congener groups. Quantitation of the C-13 labelled dioxin mixture was carried out using a reference standard mixture of unlabelled dioxins consisting of at least one isomer for each congener group. The C-13 labelled dioxin standard is highly useful for the determination of retention windows for tetra- to octa-chlorodioxins, identification of dioxins in each congener group, and calculation of the recovery of dioxins in samples that require exten...

A combination of acid-base titrations and derivation for functional group determinations of an aquatic fulvic acid

Insight into the functional group heterogeneity of an aquatic fulvic acid has been gained via carefully designed acid-base titrations in aqueous and nonaqueous media and a newly developed derivatization technique. Results of the acid-base titrations in an aqueous medium and in the presence of heavy metals (eg. Cu(II) and La(III) indicate the presence of salicylic acid-like and catechol-like moieties in the fulvic acid molecule. The total acidic capacity can be separated into contributions from a weak acid of carboxylic functionality and a still weaker acid of -OH type by acid-base titrations in a nonaqueous medium in the presence of an internal reference standard. An organic derivatization technique in conjunction with high resolution gas chromatography has facilitated the determination of the total number of hydroxyl groups in the fulvic acid sample. The results obtained from the derivatization technique are compared with those obtained from the acid-base titrations in aqueous and nonaqueous systems.

Precise measurement of vertebral bone density using computed tomography without the use of an external reference phantom

Bone density measurement by quantitative computed tomography (QCT) commonly uses an external reference phantom to decrease scan-to-scan and scanner-to-scanner variability. However, the peripheral location of these phantoms and other phantom variables is also responsible for a measurable degradation in accuracy and precision. Due to non-uniform artifacts such as beam hardening, scatter, and volume averaging, the ideal reference phantom should be as close to the target tissue as possible. This investigation developed and tested a computer program that uses paraspinal muscle and fat tissue as internal reference standards in an effort to eliminate the need for an external phantom. Because of their proximity, these internal reference tissues can be assumed to reflect more accurately the local changes in the x-ray spectra and scatter distribution at the target tissue. A user interactive computerized histogram plotting technique enabled the derivation of reproducible CT numbers for muscle, fat, and trabecular bone. Preliminary results indicate that the use of internal reference tissues with the histogram technique may improve reproducibility of scan-to-scan measurements as well as inter-scanner precision. Reproducibility studies on 165 images with intentional region-of-interest (ROI) mispositioning of 1.5, 2.5, or 3.5 mm yielded a precision of better than 1% for normals and 1% to 2% for osteoporotic patients--a twofold improvement over the precision from similar tests using the standard technique with an external reference phantom. Such improvements in precision are essential for QCT to be clinically useful as a noninvasive modality for measurement of the very small annual changes in bone mineral density.

Application of carbon-13 labelled dioxins mixture for analysis of polychlorinated dibenzo-p-dioxins in environmental samples by GC-MS

Positive identification and quantitation of polychlorinated dibenzo-p-dioxins (PCDD) in complicated environmental samples is described using C-13 labelled dioxin mixture as an internal reference standard. The C-13 labelled dioxin mixture as an internal reference standard. The C-13 labelled dioxin mixture and a municipal incinerator (MSW) flyash extract shows the same number of isomers in each tetra to octa-chloro dioxin congener groups. The C-13 labelled dioxin standard is highly useful for determination of retention windows for tetra- to octa-chlorodioxins, identification of dioxins in each congener group, and calculation of recovery of dioxins in samples that require extensive sample clean up prior to GC-MS analysis. Its application for retention time window determination and as an internal reference standard for quantitation of dioxins in MSW flyash extract and identification of dioxins in a complex sample from a PCB fire and a fish tissue is demonstrated.

Simultaneous determination of the prodrug zofenopril and its active drug in plasma by capillary gas chromatography-mass-selective detection

After oral administration of zofenopril, the active sulfhydryl angiotensin-converting enzyme inhibitor is released. Zofenopril is currently under clinical investigation as an antihypertensive. Blood samples are reacted with N-ethylmaleimide, immediately after collection, processed into plasma and stored frozen for subsequent analysis. After addition of two internal reference standards, one each for the prodrug and the active compound, the plasma samples are purified by a combination of liquid-liquid and solid-phase extractions. The dried methylated extracts are reconstituted with tetramethylbenzene and chromatographed by automated splitless injection on a fused-silica capillary column, connected to a mass-selective detector. The analytes and the internal reference standards are chromatographically resolved and a common fragment ion is monitored for the analytes. A limit of quantitation of approximately 1 ng/ml of plasma is achieved.

The study of diabetic cataractogenesis in the intact rabbit lens by deuterium NMR spectroscopy

The polyol pathway has been implicated in the process of diabetic cataractogenesis. We report the use of deuterium ( 2H) spectroscopy for dynamically monitoring the polyol and glycolytic pathways in the single intact rabbit lens. Using 2H labeled C-1 D-glucose, the formation of sorbitol from glucose and the metabolism of sorbitol to fructose was dynamically monitored at 5.5 mM and 35.5 mM glucose concentrations. The accumulation of sorbitol at 35.5 mM glucose concentration was prevented by the inhibition of aldose reductase using an inhibitor (Sorbinil). 2H spectra were obtained in short acquisition times because of the short T 1's of deuterated metabolites. A further advantage of 2H spectroscopy is that the natural abundance resonance of water (HDO) can be used as an internal reference standard. These findings confirm previous studies and demonstrate for the first time by NMR spectroscopy activity in the polyol pathway at low glucose concentrations.

Ploidy and Proliferative Activity of Human Brain Tumors

Ploidy and proliferative characteristics were estimated by flow cytometry of the nuclear DNA content of 92 human brain tumors. Samples were frozen at -20 degrees C immediately after surgery and single cell suspensions were obtained with a mechanical dissociation technique. Propidium iodide was employed for nuclear DNA staining. Human normal brain tissue was used as internal diploid reference standard. 86% of benign tumors had unimodal DNA distribution with a DNA index (DNA I = modal channel of the G0/1 peak of the studied population/modal channel of the G0/1 peak of the normal brain) usually within the diploid or near-diploid range. 14.0% had aneuploidy, with an additional cell peak having a median DNA I of 1.60. Among malignant tumors, these figures were 61.2 and 38.8% (p less than 0.001). The percentage of S phase cells was higher in malignant (median = 3.6) than in benign tumors (median = 2.0, p less than 0.01), without correlation to histological tumor subtype. Flow cytometry appears to be a useful method for evaluating differences in DNA distribution in tumors of the central nervous system.

Internal reference standards in ionic regulation and the predictability of ionic concentrations in animals

1. 1. The regulation of ions at similar concentrations in most individuals of a species suggests the existence of internal reference standards. 2. 2. Few have been identified, but many probably relate to cell membrane properties, including potentials, surface charge densities and equilibrium constants of receptor molecules. 3. 3. Solubility may sometimes determine the product [Ca 2+][CO 3 2−]. 4. 4. Reference standards must generally each relate to more than one ionic species. 5. 5. For some concentrations, including osmolality, there may be no direct reference standard.

Simultaneous microdetermination of biperiden, haloperidol, and trihexyphenidyl in plasma and its application to pharmacokinetic analysis after concomitant intravenous administration of the drugs to rabbits.

A gas chromatographic method, using a moving precolumn sample injector and a nitrogenphosphorus detector, is presented for the simultancous quantitative analysis of biperiden, haloperidol and trihexyphenidyl in plasma. The drugs were extracted from 1 ml of plasma at pH 10.0 with ether, back-extracted into an acidic solution, and reextracted with ether after alkalinization. Diazepam was used as the internal reference standard. Processed standard curves of biperiden, haloperidol, and trihexyphenidyl plasma samples were linear over the concentration range of 1.25-50 ng/ml. Recoveries were found to be constant at various spiked doses, and the coefficients of variation were less than 11% for each drug. Plasma concentration-time courses after simultaneous administration of the three drugs to rabbits were analyzed by model-independent moment analysis. The volumes of distribution at the steady state per body weight of biperiden, haloperidol, and trihexyphenidyl were determined to be 18.5, 16.7, and 17.6 1/kg, respectively. Plasma clearances of the three drugs were 89.7, 61.7, and 79.9 ml/min/kg, respectively.

Standardization of high-resolution flow cytometric DNA analysis by the simultaneous use of chicken and trout red blood cells as internal reference standards.

Determination of nuclear DNA content by flow cytometry requires comparison with a reference standard. The use of external standards such as lymphocytes or granulocytes is time-consuming and inaccurate. Chicken red blood cells (CRBC) have a DNA content of 35% of the human diploid value and have been widely used as internal standard. The ratio calculated on the basis of the peak channel numbers of the standard and the sample and used to indicate the DNA content (DNA ratio) is, however, very sensitive to changes in the zero level adjustment of the flow cytometer. If two internal standards are used the DNA ratio becomes independent of the zero level. Rainbow trout red blood cells (TRBC) have a DNA content of 80% of human diploid cells. A mixture of CRBC and TRBC was prepared and stored in small aliquots at -80 degrees C. This mixture was added to the sample before staining. The day-to-day variation of the DNA ratio obtained by use of the two standards was smaller than that obtained by CRBC alone. The possibility of sex related differences in DNA content of CRBC and TRBC was examined. The results indicated that a new batch of standards should be tested against the old batch to avoid the introduction of a systematic error.

Fourier transform infrared photoacoustic spectroscopy of pyridine adsorbed on silica-alumina and .gamma.-alumina

Relative numbers of Bronsted acid to Lewis acid sites on silica-alumina have been determined photoacoustically by an infrared analysis of chemisorbed pyridine compared to similar adsorption of ..gamma..-alumina that has only Lewis sites. Results are similar to those obtained by earlier IR transmission studies that suggested the use of framework vibrations of silica as an internal reference standard. The 20% coverage of the silica-alumina surface by pyridine adsorbed at Bronsted sites found photoacoustically is in good agreement with a previous value of 17% estimated from transmission spectroscopic data. Reproducibility of the photoacoustic measurements is excellent.