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PubMed1997

A new RNA-friendly fixative for the preservation of penaeid shrimp samples for virological detection using cDNA genomic probes.

In situ hybridization analysis of shrimp histological sections, utilizing Taura syndrome virus (TSV) specific cDNA probes, is the most sensitive diagnostic technique presently available for the detection of this penaeid shrimp viral disease. However, false negative genomic probe results are obtained frequently from samples of Pacific white shrimp, Penaeus vannamei, that have been preserved with Davidson's AFA (acetic acid, formaldehyde, alcohol) fixative and that, otherwise, demonstrate pathognomonic TSV lesions by routine histology. This problem was linked to prolonged storage of shrimp samples in Davidson's fixative, which is highly acidic (pH approximately 3.5-4). Degradation of TSV genomic RNA was hypothesized to be due to either fixative- induced acid hydrolysis and/or acidophilic endogenous ribonuclease activity. Routine H and E histology and in situ hybridization analyses were conducted on equal numbers of TSV infected P. vannamei juveniles that were preserved for four different time periods (2, 6, 10 and 14 days) with either Davidson's fixative or a new, near neutral (pH approximately 6.0-7.0), RNA-friendly fixative (R-F) that was developed by the authors. In situ hybridization assays were conducted with and without R Nase precautions and all of the samples tested contained moderate to severe TSV lesions by routine histology. Davidson's preserved samples produced weak TSV probe signals after 2 days fixation, but did not react with the probes in those samples that were stored for > 6 days in the fixative. In contrast, TSV was detectable by gene probe in all of the time treatment samples preserved with the new R-F fixative. Equivalent in situ hybridization results were obtained when the same samples were analyzed in the absence of RNase-free conditions. These findings suggest that TSV RNA is degraded when samples are stored in an acidic fixative, such as Davidson's, for more than 2 days and that this problem can be prevented through preservation of shrimp samples with R-F fixative. The efficacy of this new fixative is demonstrated and the results show that RNase-free conditions are not necessary for conducting TSV in situ hybridization analyses.

Animals↗
PubMed1999

[Usefulness of a gene amplification technique (LCx MTB) in the diagnosis of tuberculosis: preliminary results with different sputum samples].

OBJECTIVE: To evaluate the use in non-sputum samples of a commercial molecular amplification kit (LCx MTB, Abbott Diagnostica) (LCx) for the diagnosis of tuberculosis. MATERIAL AND METHOD: Ninety-nine non-sputum samples from the same number of patients (bronchoalveolar, pleural and ascitic fluid, fecal samples, blood cultures, biopsies from different sites, cerebrospinal fluid, urine and gastric juices) and 14 sputum samples (10 from patients clinically suspected of having tuberculosis and 4 from patients diagnosed of tuberculosis and undergoing appropriate treatment for at least one month). All samples were LCx processed according to the manufacturer's instructions. The reference diagnosis was obtained by the Löwestein-jensen method and when results were inconsistent, we took into account the degree of clinical suspicion, response to treatment and histology. RESULTS: Seven of the 99 samples were positive by the LCx technique, and 6 of the 7 were also LJ positive; 1 could not be evaluated because of culture contamination. One LJ positive culture was LCx negative. Only one sample was positive by Ziehl-Neelsen (ZN) staining. Ninety-two samples were LCx negative, with 91 showing no growth at all. Sensitivity was 86% and specificity 98%. Atypical mycobacteria were detected in 4 cases, all of which were LCx negative. CONCLUSIONS: Diagnosis of tuberculosis by applying the LCx system to various types of samples other than sputum is simple, rapid, sensitive and specific.

Humans↗
PubMed1998

Study of the effect of gamma-irradiation on bovine serum samples on the ability of monoclonal antibodies to detect invariant antigens of Trypanosoma congolense, T. vivax and T. brucei in enzyme-linked immunosorbent assays.

Samples of bovine serum from uninfected and African trypanosomes-infected animals were tested before and after gamma-irradiation, using three sandwich enzyme-linked immunosorbent assays (ELISA). Each test system utilized a different monoclonal antibody, reputedly allowing the specific detection of conserved-invariant cytoplasmic antigens of trypanonosomes, T. congolense, T. vivax, and T. brucei, respectively. Results have identified two groups of samples. The first contained samples where there were unequivocal ELISA results indicating positivity and negativity, for non-irradiated samples. In this group, irradiation had no effect on the diagnostic sensitivity of the assays. All samples shown to be positive before irradiation remained positive and those shown to be negative, remained negative. There was, however, a statistically significant reduction in signal in each of the ELISAs following irradiation. The second group contained samples identified before irradiation as flanking the diagnostic negative/positive threshold of OD > or =0.05. These showed a negative bias after irradiation of the order of OD -0.01, which was shown to be statistically significant by paired t-statistics. Without correction of the given diagnostic negative/positive threshold, bovine sera with OD values around the threshold were expected to deliver more false negative test results upon irradiation. This was confirmed when serological data were compared with parasitological findings; where three times more false negative test results were found from irradiated serum samples. Consequently, for this group of irradiated bovine samples tested by ELISA, the re-adjustment of the diagnostic negative/positive threshold of the ELISAs using defined irradiated serum samples is recommended; otherwise, the frequency of false negative results might be increased.

Africa, Eastern↗
PubMed2001

Classical swine fever virus (C strain) distribution in organ samples of inoculated piglets.

Enzyme linked immunosorbent assays (ELISAs) and a nested polymerase chain reaction after reverse transcription (RT-PCR) were used for the detection of the Chinese strain (C strain) of classical swine fever virus (CSFV) in blood and tissue samples of experimentally inoculated piglets. One group of 10 piglets was inoculated with C strain material from rabbits and a second one with material from infected minipig kidney (MPK) cell culture. Tested blood samples were taken on the day of inoculation as well as on days 2, 4, 6, 8, 10, 13 and 16. Samples of spleen, tonsil and brain tissue were collected from piglets on days 6, 8, 10, 13 and 16 and tested for glycoprotein E(RNS) and protein NS2-3 using commercially available ELISA kits. E(RNS) and NS2-3 were detected earlier in blood samples of piglets inoculated with the C strain propagated in a cell culture. Regardless of propagation the presence of the viral E(RNS) and NS2-3 was detected in spleen and tonsil samples simultaneously. The C strain propagated in a cell culture was found in only one brain sample, whereas, the virus propagated in rabbits was detected in 70% of the brain samples. For the detection of the CSFV RNA in blood samples, a part within the 5' non-coding region was amplified. The differences in the results gained by antigen detection in blood samples decreased when nested RT-PCR was used.

Animals↗
PubMed1997

Detection of Arcobacter species in gastric samples from swine.

Swine stomachs were surveyed for evidence of Arcobacter spp. and Helicobacter spp. infections associated with gastric ulceration. A nested PCR test targeted to the 16S rRNA was developed to detect many Arcobacter spp. and Helicobacter spp. An internal oligonucleotide probe was used for differentiation and confirmation of the PCR product. Tissue samples were obtained from the nonglandular and glandular regions of 86 swine stomachs. Evidence of infection with these microbes was detected in 51%, with 77% of the positive samples being identified as A. butzleri using a highly specific probe. Nonglandular stomach samples (44%) were more likely to be positive by PCR than samples from the glandular (23%) region. Gross lesions of any stage of gastric ulceration, ranging from parakeratosis, erosions and ulceration, were observed in 24% of stomachs examined. Of 21 samples with lesions, 52% were positive by the broadly reactive PCR assay for Arcobacter spp. and Helicobacter spp. The majority of PCR-positive samples (75%) had no gross lesions. When a single step PCR assay that was more specific for Arcobacter spp. was used on the nonglandular stomach samples, 10.4% of the 86 samples were positive. Arcobacter spp. were cultured from four of the sample stomachs. Partial sequencing of the 16S rRNA gene identified the isolates as A. butzleri (n = 2), A. cryaerophilus, (n = 1), and a mixed culture of A. butzleri and another Arcobacter spp. (n = 1). A single step PCR assay targeted to the urease gene and culturing methods were used to screen for H. pylori or other closely related urease positive bacteria, but none were found.

Animals↗
PubMed1997

Stability-indicating high-performance liquid chromatographic assay of busulfan in aqueous and plasma samples.

A sensitive, specific and stability-indicating high-performance liquid chromatographic (HPLC) assay, involving pre-column derivatization and solid-phase extraction (SPE), was developed and validated for the quantitation of busulfan (BU) in aqueous and plasma samples. The linearity of the assay was in the concentration ranges of 0.15-10 microg/ml and 0.15-3 microg/ml for aqueous and plasma samples, respectively. The within-day and between-day variations were 2.90 and 3.31%, respectively, for the aqueous samples, and 9.24 and 14.56%, respectively, for the plasma samples. The overall recovery, derivatization yield and SPE efficiency of BU from plasma samples were 82.03, 108.01 and 86.69%, respectively. Forced degraded samples, either in highly acidic, neutral or basic medium, produced no interfering peaks in the chromatogram. The reported assay requires only 0.2 ml of plasma for the analysis, and its sensitivity is 150 ng/ml by monitoring samples at a wavelength of 254 nm, sufficient to study the plasma pharmacokinetics of BU in rats after a clinically relevant oral dose. Moreover, the sensitivity of the assay can be significantly increased to 30 ng/ml by monitoring samples at a wavelength of 278 nm. The applications of the assay were demonstrated with BU solubility measurements in two aqueous systems and with plasma samples from a Sprague-Dawley rat for an in vivo pharmacokinetic study. In addition, the assay has been employed in the development of a patented intravenous formulation, and in evaluations of stability, preclinical pharmacokinetics in rats and dogs, and clinical phase I trial of the formulation. The assay is readily adaptable to clinical therapeutic drug monitoring.

Animals↗
PubMed1998

Sample pooling to expedite bioanalysis and pharmacokinetic research.

In the progression from drug discovery to development, not only pharmacokinetic (PK) characterization needed for lead compound selection often becomes a rate-limiting step, but also high volume of routine sample analysis ensued from numerous required biodisposition studies for the lead compounds and their back-ups often place a burdensome hurdle to the throughput of IND and NDA development phases. Higher throughput of PK screening via cocktail dosing has been reported to accelerate PK screening in the discovery phase. However, concerns on drug-drug interactions and other limitations associated with the cocktail M-in-One dosing (multiple compounds per dose per animal) has prompted the present investigation of sample pooling alongside One-in-One dosing strategy (one compound per dose per animal) as an alternative to the cocktail dosing approach. Using traditional HPLC for bioanalysis as an example, the present study illustrate the concept and usefulness of sample pooling that could facilitate the throughput of PK screening and characterization in both discovery and development phases. Six proprietary dopamine D4 receptor antagonist preleads representing three different chemical classes, used as model compounds (C1-C6), were administered orally to rats. One rat received one compound and three rats were used for each compound. Six unknown plasma samples from six different rats at each time point were pooled. The pooled plasma samples were extracted by a one-step liquid-liquid extraction and concentrations of the six preleads were quantitated simultaneously. By sample pooling, a substantial amount of PK information was obtained at the same time for the six preleads, which requires much less workload than when bioanalysis is dealt with one compound at a time. For the first time in one aspect of innovative bioanalysis, the present investigation has demonstrated that sample pooling following One-in-One dosing can be utilized to enhance the throughput rate in PK screening in discovery phase. The sample pooling approach is likely to be useful in enhancing the throughput of PK characterization in development phase. With the advent of LC-MS and its becoming user-friendly, where separation of drug compounds is no longer an issue, the uniqueness of sample pooling may also pose a new way of thinking in regard to the old ways of handling bioanalysis for traditional PK research.

Animals↗
PubMed1994

Clinical ramifications of posterior and anterior facial height changes between treated and untreated Class II samples.

Some clinicians have suggested that one primary difference between successful and unsuccessful Class II treatment outcomes is the relative change of anterior and posterior facial heights. Successfully treated cases are claimed to exhibit greater increases in posterior facial height (PFH, articulare to gonion) than in the anterior facial height (AFH, menton to palatal plane). This conjecture was tested here by recalling a treated Class I sample and a treated Class II sample and by comparing the differences found in these samples to an untreated Class II sample at the same ages. The PFH/AFH ratio increased significantly more in the treated Class I and Class II samples during the active phase of treatment than in the untreated Class II sample at the same ages. During the years from posttreatment to recall (mean = 6 yrs), there was a significant increase in the PFH/AFH ratio in the treated Class II sample due to a greater increase in PFH than AFH. This ratio continued to improve after all appliance therapy had been discontinued, and it did not occur in the treated Class I sample nor in the untreated Class II sample. The conclusion was that the patient with a Class II malocclusion, if treated, continues to change favorably over time. This favorable change, in turn, helps maintain the Class II correction.

Adolescent↗
PubMed1994

Identification of M. tuberculosis ribosomal RNA in mouthwash samples from patients with tuberculosis.

It is often not possible to obtain satisfactory sputum samples from patients suspected of having pulmonary tuberculosis. In this study, we asked whether it was possible to identify M. tuberculosis ribosomal RNA (rRNA) in mouthwash samples, and in a prospective clinical study, whether the presence of this rRNA correlated with clinical M. tuberculosis infection. Using a combination of reverse transcriptase and polymerase chain reaction amplification, it was possible to identify M. tuberculosis rRNA in mouthwash samples. M. tuberculosis rRNA was identified in mouthwash samples from patients with active tuberculosis more commonly than in samples from control subjects (P < 0.05). The test was positive in four of ten patients with active pulmonary tuberculosis, one of eight contacts, none of eight past cases of tuberculosis, two of eight patients with other diagnosis and one of five healthy volunteers. M. tuberculosis rRNA was identified in sputum from four of eight patients and in bronchoscopy trap samples from four of five patients with active tuberculosis. However, one of ten sputum samples and two of five bronchoscopy samples from subjects with a clinical diagnosis other than active tuberculosis were positive. These results indicate that although it is technically possible to identify M. tuberculosis on the basis of the presence of rRNA in mouthwash samples, the poor sensitivity and specificity of the technique suggest that it is unlikely to be useful clinically.

Base Sequence↗
PubMedDate not supplied

Study of the representativity of uranium and thorium assays in soil and sediment samples by alpha spectrometry.

The activity concentrations of soil and sediment samples are determined by alpha spectrometry with a sample size usually < 1 g. Hence, a systematic and careful procedure must be followed in the sampling and pre-treatment of the sample in order to ensure that the aliquot analysed represents the activity concentration of the entire sample. Statistical analyses of the activity concentrations of uranium and thorium isotopes in soil and sediment samples were performed to study the activity variation due to the lack of the representativity of the sample. These studies showed that, although some uncertainty in the determination of the sample activity concentration may be due to the lack of representativity (about 10%), the procedure followed for the pre-treatment of the sample can be considered adequate, providing an acceptable level of representativity.

Journal Article↗
PubMed2004

Hysteroscopically targeted biopsies compared with blind samplings in endometrial assessment of menopausal women taking tamoxifen for breast cancer.

STUDY OBJECTIVE: To determine the validity of tissue sampling accomplished by hysteroscopically targeted or blind biopsies in the assessment of endometrial morbidity associated with tamoxifen treatment. DESIGN: Retrospective, unrandomized study (Canadian Task Force classification II-2). SETTING: Public hospital. PATIENTS: One hundred seventy-six menopausal women who had an endometrial stripe of more than 4 mm on transvaginal ultrasonography. INTERVENTION: Review of histopathologic reports of patients undergoing hysteroscopy followed by targeted (94 samplings) or blind (82 samplings) endometrial biopsies. MEASUREMENTS AND MAIN RESULTS: Histopathology was considered the reference test to assess endometrial morbidity, and correlates with hysteroscopic findings were made to evaluate the validity of the two sampling procedures. Overall, in 23 women (13.0%) tissue samples were insufficient for pathologic evaluation. Functional or atrophic endometrium and cystic atrophy were found in 51 (28.8%) and 37 patients (21.0%), respectively. Polyps, hyperplasias, and carcinomas were found in 38 (21.5%), 19 (10.7%), and 6 (3.3%), respectively. Blind biopsies failed to detect 5 of 5 polyps and 33 of 37 cystic atrophies, and in 34.1% of cases provided insufficient tissue for diagnosis; however, no hyperplasias or carcinomas were undetected. All specimens collected under vision were pathologically evaluable; 34 of 38 hysteroscopic reports of cystic atrophy were confirmed, and neither endometrial polyps nor hyperplasias and carcinomas were undetected. In distinguishing between normal and abnormal endometrium, hysteroscopy showed sensitivity and negative predictive value of 100% regardless of sampling modality. We found better specificity (80.0% vs 68.9%) and positive predictive value (68.9% vs 43.7%) for hysteroscopic diagnosis when tissue was collected under vision compared with blind sampling. CONCLUSION: In women taking tamoxifen, endometrial evaluation performed by blind sampling is safe in excluding hyperplasias or carcinomas. For complete understanding of tamoxifen-associated morbidity, hysteroscopy with sampling under vision has better diagnostic compliance.

Aged↗
PubMed2003

Susceptibility corrections in solid state NMR experiments with oriented membrane samples. Part II: theory.

In solid state NMR analysis of oriented biomembranes the samples typically have the shape of a rectangular block, formed by stacking a number of glass slides coated with the membranes under investigation. Reference material may be provided internally in the volume of the block or as an external layer on its surface, as described in the accompanying paper [J. Magn. Reson. 164 (2003) 104-114]. The demagnetizing field resulting in such non-spheroidal samples is inhomogeneous. It shifts and broadens the NMR lines of both the sample and of the reference, as compared to the ideal of a spherical sample. The magnitude of these effects is typically of the order of a few ppm. To determine the necessary corrections, a general analysis is presented here for the demagnetizing field of a layered sample of rectangular block geometry, with the normal of the layers parallel to the main field or tilted about an axis of the block. The correction to the line position of the block sample is found to be approximately equal to that of the spheroid which can be inscribed into the block, and for which the correction is well known. For an external reference layer, placed on top of the block, the correction can be found by the same approximation, invoking a simple mirror concept. The layered structure of the block can be accounted for by using an average magnetic susceptibility. Sample and support materials contribute to that average according to their volume filling factors. If the sample material is anisotropic at the molecular level, as e.g. lipid bilayers are, the resulting anisotropy of the block is reduced by the filling factor of the sample material.

Anisotropy↗
PubMed2004

[Analysis of respiratory muscle structure and tumor necrosis and insulin-like growth factor expression in chronic obstructive pulmonary disease: are samples valid if obtained during thoracotomy performed because of localized pulmonary neoplasia?].

OBJECTIVE: Various methods have been used to obtain samples to study the structure of human respiratory muscles and the expression of diverse substances in them. Samples are most often obtained from autopsies, from muscle biopsies during thoracotomy performed because of a localized pulmonary lesion (TLL), and from ambulatory thoracoscopic biopsy in patients free of comorbidity (AT). The disadvantage of the first 2 of these methods lies in the possibility of interference from factors related to the patient's death in the first case or from the disease that necessitated surgery in the second. Although AT is free from the disadvantages of the other 2 methods, it is impossible to obtain samples of the diaphragm the principal respiratory muscle with this procedure. The objective of this study was to analyze the fibrous structure of the external intercostal muscle of patients with chronic obstructive pulmonary disease and to quantify the expression of the principal inflammatory cytokine tumor necrosis factor alpha (TNF-alpha)- and of insulin-like growth factor (IGF-1) in the same muscle, comparing the results obtained with TLL and AT samples. METHODS: Prospective and consecutive samples were taken of the external intercostal muscle (fifth space, anterior axillary line) in 15 patients with chronic obstructive pulmonary disease (mean [SD] age 66 [6] years; forced expiratory volume in 1 second 49% [9%] of predicted; PaO2 75 [9] mm Hg). Samples were taken during TLL (8 patients, all with pulmonary neoplasms but carefully selected in order to rule out systemic effects) or TA (7 patients). Patients with serious comorbidity were excluded from the second group. Samples were processed for structural analysis of fibers (immunohistochemical and enzymatic histochemical) and genetic expression of TNF-alpha and IGF-1 (real-time polymerase chain reaction). RESULTS: No differences in the structure of fibers were found between the 2 groups. No differences were observed in the expression of TNF-alpha or IGF-1. CONCLUSIONS: Using rigorous criteria, the TLL method appears to be suitable for studying the structural characteristics and expression of inflammatory cytokines and growth factors in the external intercostal muscle. Moreover, it can also be inferred that TLL is probably also useful for obtaining samples of the diaphragm, a muscle which cannot currently be sampled by any alternative method.

Aged↗
PubMed1998

Detection of canine distemper virus in 100% of Paget's disease samples by in situ-reverse transcriptase-polymerase chain reaction.

Previous evidence implicating paramyxoviruses in the aetiopathology of Paget's disease of bone has been controversial. While several groups have demonstrated the presence of paramyxoviruses using electron microscopy, immunohistochemistry, and molecular biological techniques, others have found no evidence of viruses using reverse transcriptase-polymerase chain reaction (RT-PCR). We have previously provided evidence that canine distemper virus (CDV) is present in approximately 65% of samples of pagetic bone, using in situ hybridization and RT-PCR; however, these results have been criticized. To further investigate the possible Role of CDV, we have now developed the technique of in situ-RT-PCR (IS-RT-PCR) to examine for the presence of CDV-nucleocapsid (CDV-N) ribonucleic acid (RNA) in pagetic bone. Control samples consisted of uninvolved sites from patients with the disease, normal bone, and several active remodeling states. IS-RT-PCR was optimized to detect CDV-N using distemper-infected vero cells. The specificity of the technique was confirmed using vero cells infected with CDV, which showed amplified signal following IS-RT-PCR, and cells infected with measles virus (MV), in which no positive signal for CDV was detected by IS-RT-PCR. Following conventional in situ hybridization, CDV-N was detectable in 10 of 15 pagetic bone samples. However, after five, and particularly 10, cycles of IS-RT-PCR, CDV-N was found in all 15 samples. There was no evidence of CDV in four samples from uninvolved sites from pagetic patients, or in any of the other control samples. In this study, using the novel technique of IS-RT-PCR, CDV was found to be present in 100% of pagetic samples examined. There was no evidence of the virus in any of the control samples, including samples of bone from uninvolved sites from patients with Paget's disease. These results provide additional proof that CDV is present within pagetic bone and further support the hypothesis that paramyxoviruses are involved in the etiopathology of Paget's disease.

Animals↗
PubMed2007

Haptoglobin and serum amyloid A in relation to the somatic cell count in quarter, cow composite and bulk tank milk samples.

Milk somatic cell count (SCC) is the gold standard in diagnosis of subclinical mastitis, and is also an important parameter in quality programmes of dairy cooperatives. As routine SCC analysis is usually restricted to central laboratories, much effort has been invested in the search for alternative biomarkers of mastitis and milk quality, including the presence in the milk of the acute phase proteins (APP), haptoglobin (Hp) and serum amyloid A (SAA). The aim of this study was to investigate relationships between Hp, SAA and SCC in quarter, cow composite, and bulk tank milk samples. Cows (n=165), without any clinical signs of disease or abnormalities in the milk or udder, from three different dairy farms, were used. Cow composite milk samples from all cows delivering milk at the sampling occasion were taken once in each herd. In one of the farms, representative quarter milk samples (n=103) from 26 cows were also collected. In addition, bulk tank milk samples from 96 dairy farms were included in the study. Samples were analysed for Hp, SAA and SCC, and relationships between the parameters were evaluated at quarter, cow and tank milk levels using Chi-square analysis. Milk samples were categorized according to their SCC, and the presence, or no presence, of SAA and Hp, based on the detection limits of the screening methods (0.3 mg/l and 1.0 mg/l for SAA and Hp, respectively). Hp and SAA were found in milk at quarter, cow composite and bulk tank levels. A large proportion (53%) of the animals had detectable milk concentrations of APP, and SAA was detected more frequently, and at higher concentrations than Hp, regardless of sample type. SAA was detected in as many as 82% of the bulk tank milk samples. Significant relationships were found between Hp, SAA and SCC at quarter and cow composite milk levels, but only between SAA and SCC at bulk tank milk level. Detectable levels of APP were more common at high SCC.

Animals↗
PubMed1998

Assessment of isoflavonoid concentrations in Australian bovine milk samples.

There is considerable interest in the possibility that diet-derived isoflavonoids may help in protection against a number of chronic diseases common in Western society. Based on animal studies, however, concerns have been raised that consumption of isoflavonoids by infants and young children may be undesirable. Clover contains isoflavonoids and therefore may represent, via milk, a source of isoflavonoids in the human diet. In this study the concentrations of daidzein (7, 4'-dihydroxyisoflavone), genistein (5, 7, 4'-trihydroxyisoflavone) and equol (7-hydroxy-(4'-hydroxyphenyl)chroman) were measured using HPLC in cows' milk samples obtained from 76 farms in three Australian states. In addition, concentrations were measured in samples collected from one South Australian factory both before and after pasteurization. Concentrations in all samples were found to be extremely low. The mean daidzein concentration was < 5 ng/ml. Mean genistein concentrations ranged from just detectable (approximately 2 ng/ml) in Victorian samples collected during summer to 20-30 ng/ml in samples from all states collected during spring when isoflavonoid-containing clover is most dominant in pasture. Mean equol concentrations ranged from 45 +/- 10 ng/ml in Victorian farm samples collected during summer to 293 +/- 52 ng/ml in Western Australian samples collected in spring. The mean concentrations of genistein and equol in post-pasteurization samples collected in spring were approximately double those for samples collected in autumn. Pasteurization had no effect on isoflavonoid concentrations. We conclude that the concentrations of isoflavonoids in Australian cows' milk are low and are therefore unlikely to have any pronounced biological effects in human consumers.

Animals↗
PubMed2004

Release of S(+) enantiomers in breath samples after anaesthesia with isoflurane racemate.

BACKGROUND AND OBJECTIVE: Isoflurane is a chiral volatile anaesthetic, routinely administered as racemate. It has a low metabolic rate and is mostly eliminated via respiration. In blood samples, S(+) enantiomers are found in greater proportion in the days immediately after administration of isoflurane racemate whereas the ratio in breath samples is unknown. METHODS: Breath and blood samples were drawn immediately after recovery and daily up to 19 days after operation from patients undergoing anaesthesia with isoflurane racemate. The percentage of isoflurane enantiomer was determined by gas chromatography mass spectrometry in blood and thermodesorption gas chromatography mass spectrometry in breath samples. RESULTS: In breath samples, there were significant differences in S(+) enantiomers at all time points compared to the racemate. During the early postoperative phase, the percentage of S(+) enantiomers were significantly enhanced whereas 5 days after surgery predominantly R(-) enantiomers (50.41%) were detected in the breath samples. Also in blood samples a statistical significant accumulation of the S(+) enantiomer was noted between days 1 and 5 compared to isoflurane racemate blood control. S(+) enantiomers were significantly higher in blood compared to breath samples and was most evident on the third day after surgery (51.43%). CONCLUSIONS: During the first days after application of isoflurane racemate, the percentage of S(+) enantiomers are higher in breath and blood samples of patients. We suggest that resorption and/or redistribution of enantiomers are responsible for the different kinetics of isoflurane enantiomers.

Anesthetics, Inhalation↗
PubMed2001

Selected reaction monitoring LC-MS determination of idoxifene and its pyrrolidinone metabolite in human plasma using robotic high-throughput, sequential sample injection.

The generation of large numbers of samples during early drug discovery has increased the demand for rapid and selective methods of analysis. Liquid chromatography-tandem mass spectrometry (LC-MS-MS), because of its sensitivity, selectivity, and robustness, has emerged as a powerful tool in the pharmaceutical industry for many analytical needs. This work presents a high-throughput selected reaction monitoring LC-MS bioanalytical method for the determination of idoxifene, a selective estrogen receptor modulator, and its pyrrolidinone metabolite in clinical human plasma samples. The described method uses short, small-bore columns, high flow rates, and elevated HPLC column temperatures to perform LC separations of idoxifene and its metabolite within 10 s/sample. Sequential injections were accomplished with a 215/889 multiple probe liquid handler (Gilson, Inc.), which aspirates eight samples simultaneously and performs its rinse cycle parallel to sample injection, resulting in minimum lag time between injections. This high-throughput method was applied to the determination of idoxifene and its metabolite in clinical human plasma samples. Sample preparation employed liquid/liquid extraction in the 96-well format. Method validation included determination of intra- and interassay accuracy and precision values, recovery studies, autosampler stability, and freeze-thaw stability. The LOQ obtained was 10 ng/mL for idoxifene and 30 ng/mL for the metabolite. Using idoxifene-d5 as an internal standard, idoxifene showed acceptable accuracy and precision values at QC level 1 (QC1, 15 ng/mL), level 2 (QC2, 100 ng/mL), and level 3 (QC3, 180 ng/mL) (85.0% accuracy +/- 12.0% precision, 95.1 +/- 4.9%, and 90.3 +/- 4.7%, respectively). The pyrrolidinone metabolite also showed acceptable accuracy and precision values (using no internal standard for quantitation) at QC1 (60 ng/mL), QC2 (100 ng/mL), and QC3 (180 ng/mL) (104.9 +/- 14.4%, 91.1 +/- 13.0%, and 90.8 +/- 12.2%, respectively). The validated method was applied to the analysis of 613 human clinical plasma samples. An average run time of 23 s/sample (approximately 37 min/ 96-well plate or over 3,700 sample/day) was achieved. The successful validation presented indicates that rapid methods of analysis can efficiently and reliably contribute to the fast sample turnaround required for high sample number generating processes.

Chromatography, Liquid↗