Effect of chlorhexidine on plaque development in an artificial mouth.

The effect of chlorhexidine on the development of plaque, resulting from the inoculation with saliva of a tooth mounted in an artificial mouth, has been studied. The agent delayed the formation of plaque and also inhibited changes in pH and Eh, whether sucrose was present or not. Applied as a rinse at intervals of 12 h it prevented formation of plaque and pH and Eh remained constant. Moreover, chlorhexidine inhibited pH and Eh changes in established plaque. A single application of a gel containing chlorhexidine also greatly inhibited plaque development.     

Characterization of polymicrobial biofilms obtained from saliva or carious lesions in dentin

Abstract

This study aimed to compare the formation of polymicrobial biofilms using carious dentin or saliva as inoculum for application in in vitro microbiological studies on caries research. For biofilm growth, combined samples of infected dentin or saliva from three donors were used. The biofilms were grown on glass coverslips, under a regimen of intermittent exposure (6?h day^?1) to 1% sucrose for 4?days. Total bacterial loads, as well as specific aciduric bacteria and mutans streptococci loads were quantified and correlated with biofilm acidogenicity and susceptibility to chlorhexidine. The data were evaluated using the Student’s-t, Mann Whitney and Kruskal-Wallis tests. The two biofilms showed similar microbial loads (total bacteria, aciduric bacteria and mutans streptococci) on day 4, and high acidogenicity after 48?h and were susceptible to chlorhexidine at different time intervals. In conclusion, both dentin and saliva can be used as an inoculum in in vitro studies of processes related to biofilm formation.     

Determination of ceftriaxone, a novel cephalosporin, in plasma, urine and saliva by high-performance liquid chromatography on an NH2 bonded-phase column

A high-performance liquid chromatographic method has been developed for the determination of a new cephalosporin antibiotic in plasma, urine and saliva (mixed saliva) using normal-phase technique and an NH₂ bonded-phase column. The eluent mixture was a combination of acetonitrile and an aqueous solution of ammonium carbonate. The rapid method involved precipitation of protein from fluids by means of acetonitrile followed by automatic injection of the supernatant. The detection limit was 0.4 μg/ml for plasma, 3 μg/ml for urine and 0.03 μg/ml for saliva using UV detection.     

Targeted profiling of oral bacteria in human saliva and in vitro biofilms with quantitative real-time PCR

An in vitro plaque model based on the use of human salivary bacteria and tooth-like surfaces was previously developed for studying the formation of oral biofilm and its use for pre-clinical testing of candidate antimicrobial or antiplaque agents. In this study, a quantitative Taqman PCR assay (QPCR) was developed to compare the bacterial compositions of in vitro biofilms to parent saliva samples, and to determine the relative contributions of different species in the formation of the oral biofilm. In addition, the growth inhibition of saliva-derived plaque was evaluated by chlorhexidine. With this assay, which consisted of primer/probe sets targeting either 16S rDNA sequences present in public databases or cloned ribosomal intergenic spacer region (ISR) sequences, 15 oral bacteria derived from saliva as well as those that were responsible for biofilm formation in an in vitro plaque model were rapidly identified and quantified. Among the target organisms were Actinobacillus actinomycetemcomitans, Eikenella corrodens, Fusobacterium nucleatum, Lactobacillus acidophilus, Micromonas micros, Porphyromonas gingivalis, Prevotella intermedia, Streptococcus mutans, Streptococcus sobrinus, Tannerella forsythensis, and Veillonella parvula. Primer and probe sets developed were both sensitive and specific. The relative profiles of a number of bacteria in 45-h-old biofilms were determined and, when compared to saliva samples, it was found that most of the bacteria identified in saliva also populated the in vitro plaque, including some anaerobes. Brief exposure of biofilms to chlorhexidine resulted in significant losses in viability. This new broad spectrum QPCR assay in combination with the in vitro plaque model will be of significant value in the quantitative study of the microbial composition of human saliva, saliva-derived plaque, and pre-clinical evaluation of potential antimicrobial and antiplaque molecules.     

The short-term effects of various oral care methods in dependent elderly: comparison between toothbrushing, tongue cleaning with sponge brush and wiping on oral mucous membrane by chlorhexidine

doi: 10.1111/j.1741‐2358.2011.00577.x The short‐term effects of various oral care methods in dependent elderly: comparison between toothbrushing, tongue cleaning with sponge brush and wiping on oral mucous membrane by chlorhexidine Objectives: To explore the short‐term effects from toothbrushing, tongue cleaning with sponge brush and wiping on oral mucous membrane by chlorhexidine. Background: Numerous reports have been seen in recent years proving the effectiveness of mouth cleaning with a toothbrush for the prevention of respiratory infections among the dependent elderly. However, the short‐term effects from each oral care method have not yet been clarified. Hence, an investigation was conducted by having each subject independently perform various oral care methods for five consecutive days. Materials and Methods: The subjects consisted of 12 assistance‐dependent elderly who have difficulties with tooth brushing by themselves, have 10 or more residual teeth and are not yet using plate dentures. After the pre‐intervention examination, each of the following oral care methods were performed on the same subject on an approximately three week basis: 1) Tooth brushing 2)Tongue cleaning with sponge brush 3)Wiping on oral mucous with sponge brush by chlorhexidine. Each method was performed independently, once a day for 5 consecutive days and the subjects were reexamined on the sixth day for comparative verification. Results: Consequently, toothbrushing decreased the plaque index and gingival index significantly and an improvement of oral malodour was also acknowledged (p < 0.01). Tongue cleaning with a sponge brush decreased the tongue coat score significantly (p < 0.05) and oral malodour was also improved (p < 0.01). Wiping on oral mucous with a sponge brush soaked in chlorhexidine significantly decreased opportunistic infections in the pharynx region (p < 0.05). Conclusions: It was suggested that the use of not only a toothbrush but also chlorhexidine gluconate may be indicated for dependent elderly people in whom pathogens of opportunistic infection are detected.     

Simple and rapid liquid chromatography method for determination of moxifloxacin in saliva

A high performance liquid chromatographic method for determination of moxifloxacin in human saliva was developed. The method involved deproteinisation of the sample with perchloric acid and analysis of the supernatant using a reversed-phase C18 column (150 mm) and fluorescence detection at an excitation wavelength of 290 nm and an emission wavelength of 460 nm. The assay was specific for moxifloxacin and linear from 0.25 to 10.0 μg/ml. The relative standard deviation of intra- and inter-day assays was lower than 10%. The average recovery of moxifloxacin from saliva was 101%. Due to its simplicity, the assay can be used for pharmacokinetic studies of moxifloxacin.     

Rapid determination of methadone and its major metabolite in biological fluids by gas–liquid chromatography with thermionic detection for maintenance treatment of opiate addicts

A rapid gas–liquid chromatographic assay is developed for the quantification of methadone (Mtd) and its major metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), in biological fluids of opiate addicts. After alkaline extraction from samples with lidocaine hydrochloride as internal standard, Mtd and EDDP are separated on SP-2250 column at 220°C and detected with a thermionic detector. The chromatographic time is about 6 min. The relative standard deviations (R.S.D.) of Mtd and EDDP standards are between 1.5 and 5.5%. Most drugs of abuse (morphine, codeine, narcotine, cocaine, benzoylecgonine, cocaethylene, dextropropoxyphene etc) are shown not to interfere with this technique. The method has been applied to study the levels of Mtd and EDDP metabolite in serum, saliva and urine of patients under maintenance treatment for opiate dependence. EDDP levels were found higher than those of Mtd in urine samples from four treated patients, but lower in serum and undetectable in saliva. However, Mtd concentrations were higher in saliva than in serum.     

Sanguinarine levels in biological samples by high-performance liquid chromatography

A high-performance liquid chromatographic method is presented for the analysis of the benzophenanthridine alkaloid, sanguinarine, found in plant extracts. The method is demonstrated to be applicable to analyzing samples such as saliva and gingival crevicular fluid for sanguinarine following a simple acidified methanolic extraction step. The method utilizes an ethyl silane column with acidic and basic ion-pairing reagents in the mobile phase with a limit of detection of 3 ng of sanguinarine in a sample.     

人的粪便、血浆、唾液、呼出气体中短链脂肪酸的分析

目的建立一种顶空气相色谱-串联质谱法(HS-GC/MS)快速检测人的粪便、血浆、唾液、呼出气体中短链脂肪酸(SCFAs)的方法,初步探索人的粪便、血浆、唾液、呼出气体中短链脂肪酸的相关性。方法样品无需处理直接封存于顶空进样瓶中,顶空进样;采用DB-FFAP毛细管柱(30 m×0.25 mm×0.25μm)分离;全扫描模式检测。结果人的粪便、血浆、唾液、呼出气体中均含有短链脂肪酸。在人的粪便、唾液样本中均检测到8个短链脂肪酸(乙酸、丙酸、异丁酸、丁酸、异戊酸、戊酸、异己酸、己酸);血浆、呼出气体样本中均检测到7个短链脂肪酸(未检测到异己酸)。结论初步推测人的粪便、血浆、唾液、呼出气体中的短链脂肪酸具有一定的相关性。本方法简单、快速、灵敏,可用于人的生物样品中短链脂肪酸的快速检测。     

Development and application of a direct radioimmunoassay for aldosterone in saliva

A previously described direct radioimmunoassay for plasma aldosterone has been modified to enable direct measurement of the steroid in saliva. The specificity of the method has been demonstrated by assay after high pressure liquid chromatographic purification of saliva extracts. Assay of matched plasma and saliva samples taken from normal subjects during unrestricted and controlled sodium intakes, either under basal conditions or while undergoing ACTH stimulation or dexamethasone suppression, confirms that salivary aldosterone values provide a good reflection of levels in plasma. Mean salivary aldosterone values are approximately one-third of those in plasma. Sampling immediately upon waking appears to provide reliable values for salivary aldosterone, and the potential application of this technique to the screening of hypertensive patients is discussed.     

Sensitive reversed-phase high-performance liquid chromatographic method for the determination of atevirdine and its N-desethyl metabolite in human saliva or cerebrospinal fluid using solid-phase extraction

A sensitive reversed-phase high-performance liquid chromatographic method for the determination of atevirdine and its primary metabolite in human saliva or cerebrospinal fluid using solid-phase extraction is described. Samples mixed with internal standard and sodium phosphate buffer were applied to an activated C₁₈ solid-phase extraction column. The reconstituted eluate was injected onto a Zorbax RX C₈ column utilizing a mobile phase of 100 mM ammonium acetate (pH 4.0)–isopropyl alcohol–acetonitrile (55:20:25, v/v/v). Fluorescence detection was employed with excitation at 295 nm and emission at 456 nm. Quantitation was achieved using peak-height ratios. The detection response curve was linear from 2 to 850 nM for atevirdine in both human saliva and cerebrospinal fluid and from 2 to 250 nM for the metabolite in human saliva. The method was utilized to analyze cerebrospinal fluid and saliva samples from clinical studies.     

High-performance liquid chromatographic determination of the antifungal drug fluconazole in plasma and saliva of human immunodeficiency virus-infected patients

A high-performance liquid chromatographic (HPLC) assay has been developed for the determination of the antifungal drug fluconazole in saliva and plasma of patients infected with the human immunodeficiency virus (HIV). Samples can be heated at 60°C for 30 min to inactivate the virus without loss of the analyte. The sample pretreatment involves a liquid-liquid extraction with chloroform-1-propanol (4:1, v/v). The chromatographic analysis is performed on a Lichrosorb RP-18 (5 μm) column by isocratic elution with a mobile phase of 0.01 M acetate buffer (pH 5.0)-methanol (70:30, v/v) and ultraviolet (UV) detection at 261 nm. The lower limit of is 100 ng/ml in plasma (using 500-μl samples) and 1 μg/ml in saliva (using 250-μl samples) and the method is linear up to 100 μg/ml in plasma and saliva. At a concentration of 5 μg/ml the within-day and between-day precision in plasma are 7.1 and 5.7%, respectively. In saliva the within-day and between-day precision is 10.8% (at 5 μg/ml). The methodology is now being used in pharmacokinetic studies in HIV-infected patients in our hospital.     

Determination of theophylline in serum and saliva in the presence of caffeine and its metabolites

Because of marked variability in its metabolic clearance and its narrow therapeutic range (10–20 μg/ml) investigation of each patient's clearance of theophylline is desirable. The author reports here a rapid reversed-phase high-performance liquid chromatographic (HPLC) method to determine, within 3 min, the theophylline in serum and saliva in the 0.1–50 μg/ml range. A fast HPLC column, 10 × 4.6 mm, packed with 3-μm spherical ODS packing is used with acetonitrile—methanol—buffer pH 4.7 (4:7:89) to achieve separation of theophylline from paraxanthine and matrix components. Since theophylline is a major pediatric bronchodilator, the feasibility of assay in saliva was investigated as an alternative route for determining the clearance in stressed asthmatic children. Using this method it was found that the ratio of theophylline in simultaneous serum and saliva samples is very consistent over time in the same person (± 3.99%), but inter-individually this consistency is reduced ten-fold. Simultaneous serum and saliva samples need be taken only once to obtain the ratio and the kinetics followed further with salivary samples only.     

Metabolic profiling of human saliva before and after induced physiological stress by ultra-high performance liquid chromatography–ion mobility–mass spectrometry

A method has been developed for metabolite profiling of the salivary metabolome based on protein precipitation and ultra-high performance liquid chromatography coupled with ion mobility-mass spectrometry (UHPLC–IM–MS). The developed method requires 0.5 mL of human saliva, which is easily obtainable by passive drool. Standard protocols have been established for the collection, storage and pre-treatment of saliva. The use of UHPLC allows rapid global metabolic profiling for biomarker discovery with a cycle time of 15 min. Mass spectrometry imparts the ability to analyse a diverse number of species reproducibly over a wide dynamic range, which is essential for profiling of biofluids. The combination of UHPLC with IM–MS provides an added dimension enabling complex metabolic samples to be separated on the basis of retention time, ion mobility and mass-to-charge ratio in a single chromatographic run. The developed method has been applied to targeted metabolite identification and untargeted metabolite profiling of saliva samples collected before and after exercise-induced physiological stress. δ-Valerolactam has been identified as a potential biomarker on the basis of retention time, MS/MS spectrum and ion mobility drift time.     

Specific assay for radiolabelled digoxin and its known apolar metabolites in biological fluids. I.

A specific assay method for radiolabelled digoxin and its known apolar metabolites in plasma, urine and saliva was developed. The assay permits the delineation of the pharmacokinetics of digoxin and its metabolites after single-dose administration of the drug to humans. Column chromatographic and solvent extraction procedures were used for the separation of apolar and polar compounds. Thin-layer chromatography was applied for the individual and specific assessment of digoxin and its apolar metabolites. Apolar and polar standards were used for quantitative assessments of all the procedures used. Accuracy and precision of the assay developed were evaluated in plasma, urine and saliva using biological samples spiked with known amounts of standards and by measuring replicates of biological samples obtained from pharmacokinetic studies with digoxin administration to humans.     

Determination of BAY 12-8039, a new 8-methoxyquinolone, in human body fluids by high-performance liquid chromatography with fluorescence detection using on-column focusing

A reversed-phase (RP) high-performance liquid chromatographic (HPLC) method with fluorescence detection allowing the sensitive and specific quantification of BAY 12-8039, a new antimicrobially active 8-methoxyquinolone, in biological fluids is described. The method is compared to a microbiological assay (bioassay) based on B. subtilis test strain with a limit of quantification of approximately 60 μg/l. Following dilution and centrifugation, plasma, saliva or urine supernatant is directly injected onto the HPLC system. Concentrations down to a limit of quantification of 2.5 μg/l can be quantified in plasma, saliva and urine. Data on recovery, accuracy and precision of the method throughout the whole working range as well as results on stability of the analyte are presented. The concentration data are correlated with results from the bioassay. BAY 12-8039 is stable in plasma after repeated freeze-thaw cycles and following storage at −20°C for at least 12 months. The results of HPLC measurements excellently agree with bioassay data indicating the relevance of the method as a tool in clinical development to answer pharmacokinetic questions related to antimicrobial activity. The method was applied to human plasma, saliva and urine from subjects after a single oral dose of 400 mg of BAY 12-8039.     

Analysis of methadone and metabolites in biological fluids with gas chromatography—mass spectrometry

The analysis of methadone and its metabolites in biological fluids by gas chromatography—mass spectrometry is described with deuterated methadone and metabolites as internal standards. The method allowed the determination of 20 ng methadone in 0.5 ml of plasma or saliva. Mean saliva to plasma ratio of methadone for two patients was determined to be 0.51 ± 0.13. Methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in urine were measured by selected ion monitoring. Gas chromatography—mass spectrometry was found to have advantages over conventional gas chromatographic methods in terms of ratio analysis. 1,5-Dimethyl-3,3-diphenyl-2-pyrrolidone previously reported as a metabolite was shown to result primarily from the decomposition of EDDP free base.     

Chlorpheniramine : I. Rapid quantitative analysis of chlorpheniramine in plasma,saliva and urine by high-performance liquid chromatography

A method was developed for the rapid quantitative analysis of chlorpheniramine in plasma, saliva and urine using high-performance liquid chromatography. A diethyl ether or hexane extract of the alkalinized biological samples was extracted with dilute acid which was chromatographed on a reversed-phase column using mixtures of acetonitrile and ammonium phosphate buffer as the mobile phase. Ultraviolet absorption at 254 nm was monitored for the detection and brompheniramine was employed as the internal standard for the quantitation. The effects of buffer, pH, and acetonitrile concentration in the mobile phase on the chromatographic separation were investigated. A mobile phase 20% acetonitrile in 0.0075 M phosphate buffer at a flow-rate of 2 ml/min was used for the assays of plasma and saliva samples. A similar mobile phase was used for urine samples. The drug and internal standard were eluted at retention volumes of less than 17 ml. The method can also be used to quantify two metabolites, didesmethyl- and desmethylchlorpheniramine, in the urine. The method can accurately measure chlorpheniramine levels down to 2 ng/ml in plasma or saliva using 1 ml of sample, and should be adequate for biopharmaceutical and pharmacokinetic studies. Various precautions for using the assay are discussed.     

Validation of an assay for the determination of cotinine and 3-hydroxycotinine in human saliva using automated solid-phase extraction and liquid chromatography with tandem mass spectrometric detection

The validation of a high-performance liquid chromatographic method for the simultaneous determination of low level cotinine and 3-hydroxycotinine in human saliva is reported. Analytes and deuterated internal standards were extracted from saliva samples using automated solid-phase extraction, the columns containing a hyper cross-linked styrene–divinylbenzene copolymer sorbent, and analysed by reversed-phase liquid chromatography with tandem mass spectrometric detection (LC–MS–MS). Lower limits of quantitation of 0.05 and 0.10 ng/ml for cotinine and 3-hydroxycotinine, respectively, were achieved. Intra- and inter-batch precision and accuracy values fell within ±17% for all quality control samples, with the exception of quality control samples prepared at 0.30 ng/ml for 3-hydroxycotinine (inter-day precision 21.1%). Results from the analysis of saliva samples using this assay were consistent with subjects’ self-reported environmental tobacco smoke (ETS) exposures, enhancing the applicability of cotinine as a biomarker for the assessment of low level ETS exposure.     

Validation of a cortisol enzyme immunoassay and characterization of salivary cortisol circadian rhythm in chimpanzees (Pan troglodytes)

Monitoring concentrations of stress hormones is an important tool for behavioral research and conservation for animals both in the wild and captivity. Glucocorticoids can be measured in mammals as an indicator of stress by analyzing blood, feces, urine, hair, feathers, or saliva. The advantages of using saliva for measuring cortisol concentrations are three-fold: it is minimally invasive, multiple samples can be collected from the same individual in a short timeframe, and cortisol has a relatively short response time in saliva as compared with other materials. The purpose of this study was to: (1) conduct an adrenocorticotropic hormone (ACTH) challenge as a physiological validation for an enzyme immunoassay to measure salivary cortisol in chimpanzees and (2) characterize the circadian rhythm of salivary cortisol in chimpanzees. We determined that salivary cortisol concentrations peaked 45 min following the ACTH challenge, which is similar to humans. Also, salivary cortisol concentrations peaked early in the morning and decreased throughout the day. We recommend that saliva collection may be the most effective method of measuring stress reactivity and has the potential to complement behavioral, cognitive, physiological, and welfare studies.