Ion chromatographic method for simultaneous determination of nitrate and nitrite in human saliva

A simple, rapid, accurate and sensitive method is proposed for the simultaneous determination of nitrite and nitrate in human saliva. Nitrite and nitrate present in the human saliva were determined after 10- to 100-fold dilution with ion chromatography (IC) using suppressed conductivity detection. Recoveries of nitrite and nitrate were found to be ranged between 95% and 101%. The method was linear (r2=0.9991) over the concentration working range. The detection limits were found to be 15.0 microg/l and 33.5 microg/l, for nitrite and nitrate, respectively. Ions that are present in human saliva and several other ions that are suspected to affect nitrite and nitrate determination were checked. It was found that most of the ions did not cause any interference in the determination. The method allows simultaneous determination of nitrite and nitrate in human saliva.     

Ion chromatographic determination of taurine in medicine, nutrient capsule and human urine with electrochemical detection.

A very simple and sensitive method for the determination of taurine by ion chromatography with electrochemical integrated pulsed amperometry is firstly described. Taurine was determined using 160 mmol/l NaOH as eluent and a Dionex CarboPac PA1 separation column (250x4 mm I.D.) without the interference with ten kinds of common amino acids. The peak area response of taurine was linear in the range 0.1-20 microg/ml, the detection limit was 0.034 microg/ml. The method has been applied successfully in the determination of taurine in medicinal granule, nutrient capsule and human urine. The content determined in medicinal granule is consistent with that marked by the manufacturer.     

New approaches for biocompatibility testing using cell culture

We are proposing two modified assays for biocompatibility testing which analyze the effects of cytotoxic substances leached from a biomaterial in cell culture. The biocompatibility of two vascular prostheses made of polytetrafluoroethylene was analyzed using the modified assays. One test, the "fluid medium assay" was modified by using small pieces of graft glued to a screening lid, thereby reducing the possibility of mechanical injury to cultured cells by free fragments of the tested biomaterial. Another test, the "cell inhibition assay" was modified in that the biomaterial to be tested was ground into small pieces while at very low temperature. The measure of cell toxicity used was the effect on DNA replication. Our results suggest that these modified assay methods can be used to evaluate the biocompatibility of biomaterials.     

High-performance thin-layer chromatographic determination of theophylline in plasma

A high-performance thin-layer chromatographic method for quantification of theophylline from plasma is described. The calibration curves of theophylline in methanol and in plasma were linear in the range 20-100 ng. The correlation coefficients were 0.9971+/-0.0011 and 0.9955+/-0.0003 for standard curves in methanol and in plasma, respectively. The limit of quantitation of theophylline in human plasma (assay sensitivity) was 20 ng and no interference from endogenous compounds was observed. The recovery of theophylline from human plasma using the described assay procedure was 89%. The mean relative standard deviations for intra- and inter-day analyses were 1.67% and 2.34% for 50 ng and 2.25% and 3.14% for 75 ng theophylline concentration, respectively. The method was utilized to monitor plasma concentration of theophylline post-administration of sustained release tablets in human patient volunteers.     

Regulation on the biocompatibility of bioactive titanium metals by type I collagen

To study the regulation on the biocompatibility of titanium metal surface structure, the interaction between the collagen and the titanium surface structure were studied with titanium surfaces subjected to anodic oxidation and acid-alkali treatment. The cell response on the treated surfaces was studied in vitro experiments of MG63 osteoblasts. The effects of different collagen adsorption ability on the biomineralization were investigated with simulated body fluid (SBF) experiment and osteoblasts culture experiments in a mineralization culture medium. It was found that the collagen adsorption ability was controlled by the wettability. The acid-alkali treated titanium could adsorb much more collagen on its surface. The abilities of cell attachment and proliferation were improved after collagen soaking. The apatite formation ability was inhibited in SBF after collagen adsorption on the surfaces, but improved in cell-involved situation. The ALP and OCN activity of MG63 cells assay showed the collagen on the titanium surface could enhance the bioactivity of the cells, which could accelerate the biomineralization process in cell culture experiments. The result indicated that the different adsorption ability of type I collagen could regulate the biocompatibility of titanium metal surface. It is possible to optimize the biocompatibility of the titanium metals by using suitable surface modification method.     

Extensive H(+) release by bone substitutes affects biocompatibility in vitro testing

Bone substitutes are widespread in orthopedic and trauma surgery to restore critical bony defects and/or promote local bone healing. Cell culture systems have been used for many years to screen biomaterials for their toxicity and biocompatibility. This study applies a human bone marrow cell culture system to evaluate the toxic in vitro effects of soluble components of different bone substitutes, which are already in clinical use. Different specimens of tricalcium phosphates (TCP) (Vitoss, Cerasorb), nondecalcified bovine bone (Lubboc), demineralized human bone matrices (DBM) (Grafton Flex/Putty), and collagen I/III matrix (ACI-Maix) were tested in Dulbecco's modified Eagle's medium (DMEM) and MesenCult culture solution and compared with a biomaterial-free cell culture. Biocompatibility parameters were cell viability evaluated by phase-contrast microscopy and laser flow cytometry, morphology, and the local H(+) release by bone substitutes. There were significant differences (p < 0.05) between the tested biomaterials and culture solutions. Collagen I/III, non-demineralized bovine bone, and TCP materials showed advantages for cell survival over other tested biomaterials (average values of vital cells/mL MesenCult/DMEM: Collagen I/III: 1090/1083; Vitoss: 893/483; Cerasorb: 471/523; Lubboc: 815/410; Grafton Putty: 61/44; Grafton Flex: 149/57). Especially the DBM materials lead to a significant decrease of pH, which is considered to be a major factor for cell death. DMEM culture solution supports cell survival for those bone substitutes that induce an alkaline reaction, whereas MesenCult media promotes cell vitality in biomaterials, which leads to an acidification of culture solution.     

High performance liquid chromatographic determination of paracetamol in human serum

A method has been developed for the determination of acetaminophen in human serum. Samples are extracted with ethyl acetate. Beta-hydroxyethyl theophylline is used as internal standard. The solvent is evaporated under nitrogen and the residue is analyzed by high-performance liquid chromatography on Lichrosorb RP 18. Extraction efficiency, linearity and assay precision have been determined. The method is rapid and can be easily applied to determine the plasma level of patient having taken overdose of paracetamol.     

Liquid chromatographic determination of ethyl alcohol in body fluids.

A high-performance liquid chromatographic technique for ethyl alcohol determination in body fluids is proposed. Ethyl alcohol is quantitatively converted into acetaldehyde-phenylhydrazone by oxidation in the presence of alcohol dehydrogenase, nicotinamide-adenine dinucleotide and phenylhydrazine. The derivative is suitable for reversed-phase liquid chromatography and ultraviolet detection at 276 nm. The limits of linearity, detection and quantification as well as accuracy and reproducibility were investigated in water, serum and whole blood. Analytical responses were linear within the 0.008 to 5 g/l range, and the limit of quantification was 0.02 g/l both in aqueous standard and in biological matrix assays. Mean analytical recovery of ethyl alcohol in blood serum averaged 98.2+/-4.2%, imprecision (CV%) at 0.80 g/l was 2.2%, and the limit of quantification was 0.02 g/l. Serum concentrations of persons that avoided alcoholic beverages for a week were less than the limit of quantification. Ethyl alcohol concentrations in serum and whole blood compared well with those obtained by headspace gas chromatography. This simple and reliable procedure, which was also used for a urine assay, could be suitable for validation of the screening procedures used to monitor ethanol abuse.     

Comprehensive biocompatibility testing of a new PMMA-hA bone cement versus conventional PMMA cement in vitro

For more than 50 years PMMA bone cements have been used in orthopaedic surgery. In this study attempts were made to show whether cultured human bone marrow cells (HBMC) show an osteogenetic response resulting in new bone formation, production of extracellular matrix (ECM) and cell differentiation when they were cultured onto polymerized polymethylmethacrylate (PMMA)-hydroxyapatite (HA), conventional PMMA bone cement being taken as reference. Biocompatibility parameters were collagen-I and -II synthesis, the detection of the osteoblast markers alkaline phosphatase (ALP) and osteocalcin, the number of adherent cells and the cytodifferentiation of immunocompetent cells. Cement surface structure, HA stability in culture medium and chemical element analysis of specimens were considered. Fresh marrow cells were obtained from the human femora during hip replacement. Incubation time was up to ten weeks. We used atomic forced microscopy (AFM) and scanning electron microscopy (SEM) for cement specimen analysis. Fluorescent activated cell sorter (FACS), immunohistochemical staining. SEM and light microscopy (LM) served us to judge the cellular morphology. Products of the extracellular matrix were analyzed by protein dot blot analysis, SEM energy dispersive X-ray analysis (SEM-EDX) and Ca2+/PO(4)3- detection. HA particles increased the osteogenetic potential of PMMA bone cement regarding the cellular production of collagen, alkaline phosphatase (AP), the number of osteoblasts and the cellular differentiation pattern in vitro. Both tested cements showed good biocompatibility in a human long-term bone marrow cell-culture system.     

Comprehensive biocompatibility testing of a new PMMA-HA bone cement versus conventional PMMA cement in vitro

For more than 50 years PMMA bone cements have been used in orthopaedic surgery. In this study attempts were made to show whether cultured human bone marrow cells (HBMC) show an osteogenetic response resulting in new bone formation, production of extracellular matrix (ECM) and cell differentiation when they were cultured onto polymerized polymethylmethacrylate (PMMA)-hydroxyapatite (HA), conventional PMMA bone cement being taken as reference. Biocompatibility parameters were collagen-I and -III synthesis, the detection of the osteoblast markers alkaline phosphatase (ALP) and osteocalcin, the number of adherent cells and the cytodifferentiation of immunocompetent cells. Cement surface structure, HA stability in culture medium and chemical element analysis of specimens were considered. Fresh marrow cells were obtained from the human femora during hip replacement. Incubation time was up to ten weeks. We used atomic forced microscopy (AFM) and scanning electron microscopy (SEM) for cement specimen analysis. Fluorescent activated cell sorter (FACS), immunohistochemical staining, SEM and light microscopy (LM) served us to judge the cellular morphology. Products of the extracellular matrix were analyzed by protein dot blot analysis, SEM energy dispersive X-ray analysis (SEM-EDX) and Ca²⁺/PO₄ ^3- detection. HA particles increased the osteogenetic potential of PMMA bone cement regarding the cellular production of collagen, alkaline phosphatase (AP), the number of osteoblasts and the cellular differentiation pattern in vitro. Both tested cements showed good biocompatibility in a human long-term bone marrow cell-culture system.     

High-performance liquid chromatographic determination of cotinine in urine in isocratic mode

A simple procedure for the determination of cotinine, major metabolite of nicotine in urine, is described. The assay involved a liquid-liquid extraction with dichloromethane in alkaline environment. The extract was dried at ambient temperature under a gentle stream of nitrogen. The residue was dissolved in 300 microl of mobile phase and 30 microl aliquot was injected via an automatic sampler into the liquid chromatograph and eluted with the mobile phase (10-9%, v/v methanol and acetonitrile, respectively in potassium dihydrogenphosphate buffer adjusted to pH 3.4) at a flow rate of 1 ml/min on a C8 Symmetry cartridge column (5 microm, 150 mm x 3.9 mm, Waters) at 25 degrees C. The eluate was detected at 260 nm. Internal standard was 2-phenylimidazole. Sensitive and specific, this technique was performed to test urine of diabetic patients (smokers and non-smokers) admitted in an endocrinology service. Urinary cotinine seems to be a better marker of smoking status than thiocyanates.     

High-performance liquid chromatographic method for determination of tramadol in human plasma

A modified high-performance chromatographic method using UV detection was developed for determination of tramadol concentration in human plasma. Plasma samples were extracted with ethyl acetate in a one-step liquid-liquid extraction (recovery 88.5+/-2.1%). Analysis of the extract was performed on a reversed-phase LiChrospher 60 RP-select B column with a particle size of 5 microm. The mobile phase consisted of 0.05 M KH2PO4 aqueous solution (pH 3.5) and acetonitrile in a ratio of 90:10 (v/v). Metoprolol was used as the internal standard and UV detection at 225 nm was employed. Accuracy of the assay in the concentration range examined was from 1.3 to 11.9% for the intra-day run and from 1.4 to 8.1% for the inter-day run. The precision of this method varied from 1.2 to 8.7%. The reproducibility of the method was determined to be from 0.8 to 7.2% over the six-day period. A limit of detection was 9 ng/ml at a signal-to-noise ratio of 3. This validated method was then applied to the determination of tramadol concentrations in healthy volunteers after oral administration of 100 mg of tramadol in capsules of Painlax and Tramal.     

In vivo validation of a cell culture test for biocompatibility testing of urinary catheters

Biocompatibility tests have been compared for their suitability as routine safety tests for urinary catheters. Latex catheters from five manufacturers were tested by each of the following four methods: (1) a cell culture cytotoxicity assay of catheter extracts, (2) intracutaneous injection of the extracts into rabbits, (3) intramuscular implant of catheter pieces into rabbits, (4) catheterization of sheep (mucous membrane irritation). The rabbit intracutaneous and intramuscular tests are both current pharmacopoeial methods for ascertaining the suitability of polymers for medical use. The four tests each showed a cell or tissue response ranging from no detectable change to severe damage, according to the catheter batch or brand, and they each identified the same samples as most toxic and least toxic. However, they differed in sensitivity. The sheep test and the cell culture assay discriminated between catheters of intermediate toxicity and ranked as toxic catheters not identified as such by the two pharmacopoeial tests. The sheep test most closely approximates clinical usage, but is impractical for routine use. The cell culture assay is a suitable alternative. It also has the advantages of a clearly defined endpoint, good sensitivity, reproducibility, speed, and reduced animal usage.     

Rapid high-performance liquid chromatographic determination of serum gabapentin.

Gabapentin (GBP) is a new antiepileptic drug approved for clinical treatment of partial seizures in the USA. Serum GBP concentrations in 283 patients were studied using high-performance liquid chromatography with fluorescence detection. The standard curves were linear over a range of 60 ng to 15 microg/ml. The coefficient of variations were 3.4 to 8.8% and 1.4 to 9.8% for intra- and inter-assay studies, respectively. The lower limit of quantitation was 10 ng/ml. Of the 283 patients studied, 72.5% had GBP levels between 2 and 10 microg/ml, 14.8% were below 2 microg/ml and 12.7% above 10 microg/ml. The mean+/-S.E. of GBP in 283 patients was 5.38+/-0.23 microg/ml. Peak concentrations of more than 15 microg/ml and trough levels as low as 0.1 microg/ml were not uncommon. The method described was rapid, simple, highly sensitive and reproducible. Other antiepileptic drugs and endogenous compounds did not interfere with the assay.     

An in vitro model for testing biocompatibility of endodontic materials to bone

Calvaria from 6-day old mice labelled 4 days previously with ⁴⁵CaCI₂ were divided into test and control halves and each half cultured separately in vitro. Eluents from four endodontic materials, endomethasone, zinc oxide/eugenol, AH26 and gutta-percha were added separately to the culture medium of each test half. After 24 and 48 h culturing periods, the ⁴⁵CaCI₂ in the media and calvaria was measured by a standard liquid scintillation counting technique and a resorption ratio between test and control halves was computed. This ratio, based on cell mediated resorption, was an indication of toxicity of soluble components of each endodontic material. In accordance with the literature, endomethasone was found, with our method, to be the most toxic and gutta-percha the least toxic of the materials tested. This shows that our model can be used to test the toxicity of other biomaterials to bone cells in vitro.     

Ion leaching from dental ceramics during static in vitro corrosion testing

Dental ceramics are often called inert materials. It can be hypothesized, however, that differences in the composition, microstructure, and environmental conditions will affect the degree of corrosion degradation in an aqueous environment. The aims of the study were, therefore, to study the ion dissolution from glass-phase ceramics, with or without crystalline inclusions, and from all-crystalline ceramics and to compare the effects of different corrosion media. Ceramic specimens were produced from glass-phase and oxide ceramics and given an equivalent surface smoothness, after which they were subjected to in vitro corrosion (Milli-Q water at 37 +/- 2 degrees C for 18 h and 4% acetic acid solution at 80 +/- 2 degrees C for 18 h, respectively). The temperature of the corrosion solution was slowly increased until it reached 80 +/- 2 degrees C to reduce the risk of microcrack formation at the surface. The analyses of ion leakage were performed with inductively coupled plasma optical emission spectroscopy. A large number of inorganic elements leached out from the various dental ceramics. The major leaching elements were sodium and potassium; in the acid-corrosion experiments, there were also magnesium, silicon, and aluminum and, on a lower scale, yttrium, calcium, and chromium. The various glass-phase ceramics displayed significant differences in ion leakage and significantly higher leakage values than all-crystalline alumina and zirconia ceramics. No significant difference in dissolution was found between high and low-sintering glass-phase ceramics or between glass-phase ceramics with high volume fractions of crystallites in the glass phase in comparison with those with lower crystalline content. It can be concluded, therefore, that none of the dental ceramics studied are chemically inert in an aqueous environment.     

High-performance liquid chromatographic analysis for the determination of domperidone in human plasma

A specific and sensitive high-performance liquid chromatographic method for the determination of domperidone in human plasma is described. Domperidone was isolated by solid-phase extraction using nitrile SPE cartridges. The drug was eluted with a mixture consisting of methanol-triethylamine-acetic acid, separated on a reversed-phase column, and measured by fluorimetric detection after post-column photoderivatization. The absolute extraction recovery from plasma samples was 83%. The limit of quantitation was established as 1 ng/ml. The relative standard deviation of the determination of plasma levels by this method over the standard curve concentration range was less than 10%, expect with the concentration of 1 ng/ml. The suitability of the method is shown for pharmacokinetic studies.