Analytical performance of a candidate reference measurement procedure for serum 17α-hydroxyprogesterone based on liquid chromatography tandem mass spectrometry / 中华检验医学杂志

Objective:To determine the analytical performance of a candidate reference measurement procedure for 17α-hydroxyprogesterone in human serum by liquid chromatography-tandem mass spectrometry (LC-MS/MS).Methods:The serum spiked with a deuterium-labeled internal standard was extracted from serum from individual undergoing physical examination by liquid-liquid extraction with n-hexane/ethyl acetate (3∶2, v/v), separated by C18 reversed-phase chromatography and detected by positive electrospray ionization mass spectrometry. According to the Clinical and Laboratory Standards Institute (CLSI) C62-A documents, the analytical performance including linearity, limit of detection,limit of quantitation,relative matrix effect,precision and trueness,carry-over and specificity was evaluated.Results:The linear range of 17α-hydroxyprogesterone by LC-MS/MS was 0.21-119.67 μg/L. The limit of detection and limit of quantitation were 5.218 ng/L and 0.116 μg/L. The relative matrix effects were -0.02%, -0.40% and -0.90% for sera and solution mixtures in 3 different ratios (50∶50, 80:20 and 20∶80). The coefficients of variation ( CVs) of intra-assay were 1.73%-2.11%, 0.98%-1.71%, 0.47%-0.87% at 0.164 μg/L, 14.81 μg/L, 81.63 μg/L and the CVs of inter-assay were 1.82%, 1.03%, 0.80% at above three concentrations. The average recovery rates of 3 levels (0.5, 20 and 100 μg/L) were 100.4%, 101.7%, 102.8%, respectively. The measured values of GBW09829 of National Institute of Metrology were within the specified uncertainty range. Conclusion:The candidate reference measurement procedure for 17α-hydroxyprogesterone in human by LC-MS/MS is established with good accuracy and precision, which can be clinically used for measurement traceability.

Simultaneous determination of 15 mycotoxins in peanuts by ultra high performance liquid chromatography-tandem mass spectrometry with QuEChERS EMR-Lipid approach and stable isotope dilution / 上海预防医学

Objective To develop a method for the simultaneous determination of 15mycotoxins in peanuts by ultra high performance liquid chromatography-tandem mass spectrometry with QuEChERS EMR-Lipid approach and stable isotope dilution. Methods The samples were extracted by 2% formic acid acetonitrile-water (50 : 50, V/V) and then purified with QuEChERS EMR-Lipid approach.The mycotoxins were fully separated on a pentafluorophenyl column under a gradient elution with methonal-0.01%formic acid aqueous solution.The mycotoxins were analyzed by UPLC-MS/MS with multiple reaction monitoring (MRM) mode and quantified by isotope internal standard method. Results Fifteen mycotoxins had good linear relationship in the certain correlation ranges with the correlation coefficients all above 0.995 and the detection limits were 0.1－10 μg/kg.The mean recoveries ranged from 81.2% to 115.3% with RSD (n=6) varying from 2.1% to 10.7%. Conclusion The method is simple, highly sensitive, practical, and proves to be suitable for quantitative analysis of 15 mycotoxins in peanuts.

Optimized Tube Dilution Technique and Sole Carbon Utilization Assay for Anti-leptospiral In Vitro Screening of Plant Extracts

Introduction@#Leptospirosis is one of the neglected reemerging zoonoses that is of public health concern globally. The need to discover novel therapeutic alternatives for leptospirosis through screening for and elucidating the mechanism/s of the anti-leptospiral activity of plant extracts is therefore necessary. This study analyzes the optimized tube dilution technique and the BiologTM sole carbon utilization phenotype microarray as screening tool for anti-leptospiral activity of plant extracts. @*Methods@#The suitability of the optimized tube dilution technique was evaluated by determining the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and motility inhibition property of a plant extract and an antimicrobial control (pen G) against 4 dominantly circulating Leptospira serovars/serogroup in the Philippines. Likewise, the suitability of the BiologTM sole carbon utilization assay was evaluated using a plant extract and selected antimicrobials against L. interrogans serovar Manilae strain K64 and L. interrogans serovar Losbanos strain K37. @*Results@#The MIC, MBC, and motility inhibition property of a plant extract and the antibiotic controls as well as its effect on the carbon utilization phenome of the Leptospira serovars gave consistent results, within and between several runs. With standard deviation = 0 for all serovars. The MIC and MBC of the antimicrobial control (pen G), the positive control, was 10 ug/ml. The growth control (leptospires without treatment), the negative control, showed presence of motile leptospires. The MIC and the MBC of the test plant extract was 250 ug/ml – 500 ug/ml. Results of the carbon utilization phenome or pattern of carbon utilization were consistent within the 3 replicates and between two runs. @*Conclusion@#The optimized tube dilution technique and the BiologTM sole carbon utilization assay is a potential in vitro screening tool for determining anti-leptospiral activity of plant extracts.

A Fast Analytical Approach for Determination of 16 Kinds of Mycotoxins in Vegetable Oils Using Stable Isotope Dilution and Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry / 分析化学

A fast, simple and cost-effective UPLC-MS / MS method was established for determination of 16 kinds of mycotoxins in vegetable oils with stable isotope dilution technique. Samples were extracted by acetonitrile-water-acetic acid 84: 15: 1(V/ V) and then diluted using water without any further clean-up steps. The mycotoxins were fully separated on a pentafluorophenyl column. Matrix effects were efficiently compensated by the [ 13 C]-labelled internal standards. The mean recoveries at three different concentration levels ranged from 74. 2％ to 105. 6％ , with RSD varied from 0. 3％ to 13. 9％ . Finally, the method was applied to analyze several kinds of vegetable oil samples. The method was simple, rapid, high sensitive and suitable for the determination of mycotoxins in vegetable oils.

Acupressure versus dilution of fentanyl to reduce incidence of fentanyl-induced cough in female cancer patients: a prospective randomized controlled study / 대한마취과학회지

BACKGROUND: Fentanyl-induced cough (FIC) is a transient condition with a reported incidence of 18% to 65% depending on the dose and route of administration of fentanyl. Nonpharmacological methods to prevent FIC are more cost-effective than medications. Dilution of fentanyl has a proven role in the prevention of FIC. Acupressure can also prevent FIC because it has a proven role in the treatment of cough. METHODS: This study included 225 female patients with an American Society of Anesthesiologists physical status of I or II who were randomly divided into 3 groups of 75 patients each. Patients in the control group received undiluted fentanyl at 3 µg/kg, patients in the acupressure group received undiluted fentanyl at 3 µg/kg with acupressure, and patients in the dilution group received diluted fentanyl at 3 µg/kg. Coughing was noted within 2 min of fentanyl administration. The severity of FIC was graded as mild (1-2 coughs), moderate (3-4 coughs), or severe (≥5 coughs). The timing of coughs was also noted. RESULTS: The incidence of FIC was 12.7% in the control group, 6.8% in the dilution group, and 1.3% in the acupressure group. The difference in the incidence of cough was statistically significant (P = 0.008) between the control and acupressure groups. The difference in the severity of cough among the groups was not statistically significant. The median onset time of cough among all groups was 9 to 12 seconds. CONCLUSIONS: The application of acupressure prior to administration of fentanyl significantly reduces the incidence of FIC. Dilution of fentanyl also reduces the incidence of FIC, but the difference is not statistically significant.

Utilidad de las técnicas nucleares en nutrición: evaluación de la masa grasa corporal y de la ingesta de leche materna / Nuclear techniques in nutrition: assessment of body fat and intake of human milk in breast-fed infants

Las técnicas nucleares aplicables al área de nutrición son inocuas dado que utilizan isótopos estables. En particular, la dilución isotópica con deuterio evalúa la composición corporal y la ingesta de leche materna. Es un método de referencia para conocer grasa corporal y validar herramientas de bajo costo debido a su exactitud, facilidad de aplicación y por la base de antecedentes de su utilización en adultos y niños. Es no invasiva ya que utiliza saliva como muestra de análisis, lo que la hace aplicable a poblaciones pediátricas. Modificaciones en la grasa se asocian a enfermedades no transmisibles y existen individuos con peso normal pero mayor depósito de grasa. Por ello, es de utilidad analizar sus cambios como instrumento de evaluación en clínica y en programas de salud. Además, esta técnica es la forma exacta de determinar si los lactantes son amamantados exclusivamente mediante seno materno, establecer lactancia materna exclusiva y validar métodos convencionales basados en encuestas a las madres.

The application of nuclear techniques in the area of nutrition is safe because they use stable isotopes. The deuterium dilution method is used in body composition and human milk intake analysis. It is a reference method for body fat and validates inexpensive tools because of its accuracy, simplicity of application in individuals and population and the background of its usefulness in adults and children as an evaluation tool in clinical and health programs. It is a non-invasive technique as it uses saliva, which facilitates the assessment in pediatric populations. Changes in body fat are associated with non-communicable diseases; moreover, normal weight individuals with high fat deposition were reported. Furthermore, this technique is the only accurate way to determine whether infants are exclusively breast-fed and validate conventional methods based on surveys to mothers.

Autoantibodies with Mimicking Specificity Detected by the Dilution Technique in Patients with Warm Autoantibodies

BACKGROUND: The aim of this study was to investigate the frequency of autoantibodies with mimicking specificity by using the dilution technique, to assess the usefulness of the combination of the dilution technique and red blood cell (RBC) phenotyping, and to establish a pre-transfusion testing algorithm in patients with warm autoantibodies. METHODS: Serum samples from 71 patients with warm autoantibodies were tested using the dilution technique. Among them, 25 samples were adsorbed with allogeneic ZZAP (a combination of dithiothreitol and enzyme) or polyethylene glycol (PEG) and their RBC phenotypes were determined. Thirty-nine patients were transfused with our pre-transfusion testing algorithm using a combination of dilution technique and RBC phenotyping. RESULTS: Autoantibodies with mimicking specificity were detected by the dilution technique in 26.8% (19/71) of the patients and most of them were directed against Rh system antigens. The agreement of the results obtained with the dilution technique in combination with RBC phenotyping and those from ZZAP or PEG adsorption was 100% (18/18) in patients who have autoantibodies with mimicking specificity and/or alloantibodies. No clinical symptoms indicating severe acute or delayed hemolytic transfusion reactions were reported in the 39 patients transfused with our pre-transfusion testing algorithm. CONCLUSIONS: Autoantibodies with mimicking specificity detected by the dilution technique in patients with warm autoantibodies are relatively frequent, can be discriminated from alloantibodies by employing a combination of dilution technique and RBC phenotyping, and might not appear to cause severe acute or delayed hemolytic transfusion reactions.

In-Vitro Antimicrobial Activity of Membrane-Acting Antibiotics Action Against Streptococci.

Streptococcus equi subS. equi a group C streptococcus is a gram-positive bacterium and is the causative agent of one of the most prevalent diseases of horses known as strangles, although, a live attenuated S. equi vaccine for prevention of the strangles has been introduced, there are presently still no proven vaccine against S. equi. In the effort to develop an effective vaccine against S. equi, the minimum inhibitory concentrations of antimicrobials acting bacterial membrane - triclosan and 4-Methylumbelliferone (4-MU) on S. equi have been investigated. These were determined by serial dilution technique on three strains of the equine S. equi: 4047, K3, and NCTC 9682. In addition, sensitive disk test on solid media were also carried out. The result of this study demonstrated that the three strains of S. equi are very sensitive to triclosan as they inhibit certain membrane functions. However, these strains are very resistant to 4-MU. Further research to investigate the mechanism of interaction between these antimicrobial agents and the bacterial membrane components is required.

Screening, isolation and purification of antibacterial agents from marine actinomycetes.

As marine environmental conditions are extremely different from terrestrial ones, it is surmised that marine actino-mycetes might produce novel bioactive compounds. Hence marine sediments, collected from the coastal areas of Gokharna and Muradeshwara of Karnataka state, were screened Seventeen isolates were obtained on starch-casein agar media by soil dilution technique. However, only six isolates namely ACT-A2, ACT-A3, ACT-A4, ACT-A5, ACT-A7 and ACT-A15 showed significant antibacterial activity against both gram-positive and gram-negative bacteria. Morphological, cultural and biochemical characterization indicated that the isolates belong to Streptomyces genus of Actinomycetes. Further studies were carried out with the most active ACT-A2. Optimization of media, temperature and pH by shake flask fermentation indicated starch-casein, 28°C and 7 to be suitable for ACT-A2. The production of antibiotics began after 24 h, reached maximum at 72 h and maintained at the same level up to 120 h. Ethyl acetate was used to extract antibacterial compounds from the culture filtrate. TLC was done on silica gel using ethyl acetate: methanol (6:4) and direct bioautography has shown the presence of two active substance, one with Rf 0.8 has more activity than the other with Rf 0.4. Further purification is done by column chromatography using a mixture of dicholoromethane and ethyl acetate. The findings from this investigation reveal that the strain ACT-A2 in order exhibited superior antimicrobial activities to other sediment isolates of actinomycetes.

Screening, isolation and purification of antibacterial agents from marine actinomycetes.

As marine environmental conditions are extremely different from terrestrial ones, it is surmised that marine actino-mycetes might produce novel bioactive compounds. Hence marine sediments, collected from the coastal areas of Gokharna and Muradeshwara of Karnataka state, were screened Seventeen isolates were obtained on starch-casein agar media by soil dilution technique. However, only six isolates namely ACT-A2, ACT-A3, ACT-A4, ACT-A5, ACT-A7 and ACT-A15 showed significant antibacterial activity against both gram-positive and gram-negative bacteria. Morphological, cultural and biochemical characterization indicated that the isolates belong to Streptomyces genus of Actinomycetes. Further studies were carried out with the most active ACT-A2. Optimization of media, temperature and pH by shake flask fermentation indicated starch-casein, 28°C and 7 to be suitable for ACT-A2. The production of antibiotics began after 24 h, reached maximum at 72 h and maintained at the same level up to 120 h. Ethyl acetate was used to extract antibacterial compounds from the culture filtrate. TLC was done on silica gel using ethyl acetate: methanol (6:4) and direct bioautography has shown the presence of two active substance, one with Rf 0.8 has more activity than the other with Rf 0.4. Further purification is done by column chromatography using a mixture of dicholoromethane and ethyl acetate. The findings from this investigation reveal that the strain ACT-A2 in order exhibited superior antimicrobial activities to other sediment isolates of actinomycetes.

Glucose Pump Technique is as Good as Ultrasound Dilution Technique for Vascular Access Surveillance in Hemodialysis Patients / 대한신장학회지

PURPOSE: Vascular access flow (Qa) measurements are important in the surveillance protocol of hemodialysis vascular access stenosis. The glucose pump technique (GPT) is a technique for Qa measurement based on the dilution technique of a constant glucose infusion that was introduced in 1995. The aim of this study is to verify the clinical efficacy of GPT in vascular access surveillance compared with ultrasound dilution technique. METHODS: In 31 chronic hemodialysis patients with a AV fistula, we compared Qa measurements performed with GPT in pre-dialysis and the ultrasound dilution technique (Transonic HD01, Transonic System Inc., Ithaca, NY) during hemodialysis. RESULTS: Mean Qa was 1171 ml/min by GPT versus 1028 ml/min by HD01 (p=0.262). There was a strong linear correlation between the two methods (r=0.61; p<0.01). Ultrasound dilution technique HD01 yielded 6 cases of high risk Qa measurements and GPT yielded 4 cases of high risk Qa measurements. The diagnostic accuracy of GPT tested with the ROC curve was similar with ultrasound dilution technique HD01. The specificity and sensitivity was 80% and 66.7% according to Qa=600 ml/min. But sensitivity was 83.3% according to Qa=750 ml/min. CONCLUSION: GPT offers the advantage of a simple and economic bedside procedure easily performed before dialysis and had a similar diagnostic accuracy and efficiency compared with ultrasound dilution technique HD01.

Clinical Utility of Access Blood Flow Measurement by Ultrasound Dilution in Hemodialysis Patients / 대한신장학회잡지

BACKGROUND: Prophylactic stenosis correction is important in the durability of the vascular access, and routine surveillance for the detection of stenosis has been widely recommended to allow preemptive intervention before thrombotic occlusion. The ultrasound dilution technique has been considering not only a popular and validated in vascular access surveillance, but also useful in the measurement of access recirculation and cardiac output. METHODS: Access flow, recirculation, and cardiac output of seventy-six hemodialysis patients with arteriovenous fistulae were measured by ultrasound dilution technique, followed by access flow measurement by color doppler ultrasound in sixty-four patients. Access stenosis was defined as 50% and more reduction in the diameter of vessels by color doppler ultrasound. Cardiac output of thirty-seven patients by echocardiography within a year was also analyzed. RESULTS: Access stenosis was diagnosed in six of sixty-four patients by color doppler ultrasound (9.4%). Access flow in the group with stenosis (621+/-322 mL/min) was lower than the group without stenosis (1, 005+/-633 mL/min), but no statistical significance was found. The access flow measured by ultrasound dilution technique was significantly correlated with color doppler ultrasound (r=0.436, p= 0.01). The cardiac output measured by ultrasound dilution technique was also significantly correlated with echocardiography (r=0.660, p=0.01). CONCLUSION: The data did not provide statistical significances for the detection of stenosis by ultrasound dilution technique. But this study suggests that ultrasound dilution technique is a useful method in the measurement of access flow, cardiac output and recirculation. We hope further prospective studies based on our data will be performed.