Study of dried blood spots technique for the determination of dextromethorphan and its metabolite dextrorphan in human whole blood by LC–MS/MS

Dried blood spots (DBSs) technology was evaluated in an assay for the quantitation of dextromethorphan (DM) and its metabolite, dextrorphan (DT), in human whole blood using high performance liquid chromatography with tandem mass spectrometry method (LC–MS/MS). Both the parent drug and metabolite were spiked in the blood matrix and subsequently allowed to dry on a specimen collection card. The dried blood spots were removed using a manual punch and then extracted into methyl tert-butyl ether (MTBE). The organic supernatant was transferred and evaporated and the residue was reconstituted in 20% acetonitrile. The overall method recovery of DM and DT was 87.8% and 95.4%, respectively. The assay was linear over the concentration range of 0.2–200 ng/mL for both analytes. Several factors that potentially affect DBS assay quantitation were investigated, such as punch size, DBS sample punch-out location, and the volume of the blood sample pipetted on the specimen collection cards. The study determined that punch size does not affect assay quantitation accuracy. Indeed, a larger punch size increases the sensitivity due to the larger sampled blood spots. Sampling from different location on the specimen collection cards shows no significant variation for both drugs. The study also shows that acceptable results can be achieved with some variation of the sample volume, which allows a simple blood sampling procedure at the test sites. To achieve the similar lower limit of quantitation (LLOQ) as the plasma assay, several blood spots at the same concentration level were stacked together and extracted. Bioanalytical assays using the DBS technique are promising given the advantages of the method over the plasma assay.     

Immunosuppressive therapy of cyclosporin A for severe benzene-induced haematopoietic disorders and a 6-month follow-up

Long-term exposure to benzene can potentially result in severe haematotoxicities, including pancytopaenia, aplastic anaemia and myelodysplastic syndrome, which are often accompanied by life-threatening symptoms and high mortality. Previous studies demonstrate that benzene-induced haematotoxicities are immune-mediated and that cyclosporin A is a prominent treatment in acquired aplastic anaemia. This study aims to evaluate the potential role of cyclosporin A immunosuppressive therapy for severe benzene-induced haematotoxicity. Between January 2002 and December 2008, 41 patients with severe benzene-induced haematopoietic disorders from five hospitals were enrolled in the study, 22 patients received cyclosporin A, supportive treatments and/or oral testosterone undecanoate, 19 patients were treated with supportive treatments and/or oral testosterone undecanoate as the control group, and a 6-month follow-up was conducted. The results showed that in the cyclosporin A group, 19 of 22 patients (86.36%) had responded to the treatments completely or partially with increased platelets, white blood cells and hemoglobulin counts by the fourth week (P = 0.005), the sixth week (P = 0.001) and the third month post-treatment (P = 0.034), respectively. However, in the control group treated by supportive methods, only 5 of 19 patients (26.32%) responded to the treatments partially (P < 0.001). Cyclosporin A in conjunction with supportive treatments may be an effective treatment modality for patients with severe benzene-induced haematopoietic disorders, which in turn implies that these haematotoxicities are immune-mediated.     

Detection of landiolol using high-performance liquid chromatography/fluorescence: A blood esterase-sensitive ultra-short-acting β1-receptor antagonist

Landiolol hydrochloride, a new adrenergic β₁-selective antagonist having an ultra-short half-life, is used to prevent tachyarrhythmia during surgery. Since landiolol is thought to be rapidly hydrolyzed to an inactivate metabolite by esterases, quantification of the drug concentration in the blood is impractical. The landiolol concentration in blood was halved within 5 min after blood sampling. This degradation was effectively prevented by pre-treatment with neostigmine (100 μg) in the sampling tube, but not by EDTA pre-treatment, indicating that landiolol could be metabolized by pseudocholinesterase in plasma. After the one-step solid-phase extraction, fluorescence detection of landiolol reduced chromatographic background signals and then improved assay sensitivity to the lower limit of 10 ng/ml in blood; this reproducible approach yielded coefficient variation of less than 6%. The blood concentration-time profile of landiolol hydrochloride in patients of the present investigation afforded more practical assessment than previously reported studies, thus improving accuracy and facilitating detailed pharmacokinetic study in relation to the pharmacological action of drug.     

An in vivo microdialysis measurement of harpagoside in rat blood and bile for predicting hepatobiliary excretion and its interaction with cyclosporin A and verapamil

Harpagoside, a major bioactive iridoid glucoside in genus Scrophularia, has been widely used in clinical practice for the treatment of pain in the joints and lower back for its neuroprotective and anti-inflammation activities. To investigate the pharmacokinetics and hepatobiliary excretion, an in vivo microdialysis method coupled with high performance liquid chromatography was developed to monitor the concentration of harpagoside in blood and bile. The harpagoside bile-to-blood distribution ratio (AUC_bile/AUC_blood) up to 986.28 ± 78.46 significantly decreased to 6.41 ± 0.56 or 221.20 ± 18.92 after co-administration of cyclosporin A or verapamil. The results indicated that harpagoside went through concentrative elimination from the bile which was probably regulated by P-glucoprotein, providing possible clinical trials of co-administration of transporter inhibitors to decrease drug efflux, thus to enhance the curative effects.     

Application of liquid chromatography–mass spectrometry to measure short chain fatty acids in blood

A new liquid chromatography–mass spectrometry method is described to determine concentrations of the short chain fatty acids acetic acid, propionic acid and butyric acid (SCFAs) in human blood plasma. The method is based on reversed phase chromatography followed by post-column neutralization of the mobile phase with ammonia and a consecutive measurement of the SCFAs ammonia adducts using negative electro spray ionization. Sample preparation involved simple organic acid deproteinization, resulting in 100% recovery. SCFAs eluted baseline separated within a 25 min run cycle. A linear response was obtained in the range between 0 and 250 μmol/l (R² ranged from 0.997 to 0.9999). The limit of detection ranged from 0.05 μmol/l for propionic and butyric acid and 0.1 μmol/l for acetic acid. The method was tested by analyzing plasma of arterial blood, from portal vein and hepatic vein blood from patients undergoing a pylorus-preserving pancreaticoduodenectomy. As expected, the highest SCFA concentrations were found in portal plasma, hepatic vein levels were in between, while arterial concentrations were lowest. This newly developed method is suitable to determine SCFA concentrations in human plasma samples.     

Microencapsulating aerial conidia of Trichoderma harzianum through spray drying at elevated temperatures

Conidia of Trichoderma harzianum produced from either solid or liquid fermentation must be dried to prevent spoilage by microbial contamination, and to induce dormancy for formulation development and prolonged self-life. Drying conidia of Trichoderma spp. in large scale production remains the major constraint because conidia lose viability during the drying process at elevated temperatures. Moreover, caking must be avoided during drying because heat generated by milling conidial chunks will kill conidia. It is ideal to dry conidia into a flow-able powder for further formulation development. A method was developed for microencapsulation of Trichoderma conidia with sugar through spray drying. Microencapsulation with sugars, such as sucrose, molasses or glycerol, significantly (P < 0.05) increased the survival percentages of conidia after drying. Microencapsulation of conidia with 2% sucrose solution resulted in the highest survival percentage when compared with other sucrose concentrations and had about 7.5 × 10¹⁰ cfu in each gram of dried conidia, and 3.4 mg of sucrose added to each gram of dried conidia. The optimal inlet/outlet temperature setting was 60/31 °C for spray drying and microencapsulation. The particle size of microencapsulated conidia balls ranged from 10 to 25 μm. The spray dried biomass of T. harzianum was a flow-able powder with over 99% conidia, which could be used in a variety of formulation developments from seed coatings to sprayable formulations.     

A new specially designed needle significantly increases sample yield during fine needle aspiration of breast lesions

A large and increasing number of women in the western world will at some point during their life be investigated morphologically for breast cancer. Fine needle aspiration (FNA) is one morphological method which is considered to be the fastest, cheapest and the most patient-friendly approach. Nevertheless, the technique has not gained major clinical success outside of Scandinavia, mainly because of a high frequency of insufficient samples. With this in mind it is quite peculiar that standard needles which are mainly configured for blood sampling and infusion therapy, comprising large quantities of residual spaces, are used. In this study we have developed and tested a new needle dedicated for FNA, which is intended to abate this drawback by increasing the sampling yield by changing the tip angle, the cannula wall-thickness and the storage compartment. In total, 499 consecutive aspiration procedures of palpable breast lesions were performed to compare the new needle (outer diameter 0.6 mm) with standard needles (outer diameters 0.6 mm and 0.7 mm). The new needle provided three times more material than did standard needles of the same diameter. Surprisingly, the new needle also provided more material than the standard 0.7 mm needle, which increased up to almost twice the material in cases with no material in the syringe. The frequency of tests with sparse harvested material (<4 mg) was less with the new needle (9%) compared to its standard counterpart (35%). The presented results were obtained by a very skilled sampling operator. Thus for the average sampling operator who probably obtains more samples in the spare range, the new dedicated FNA needle should have even more added value.     

Use of dried blood spots and inductively coupled plasma mass spectrometry for multi-element determination in blood

The paper describes the development of an inductively coupled plasma mass spectrometry (ICP MS) method for multitrace element determination in dried blood spots (DBSs). The analytical conditions were optimized using Seronorm™ L-3 and L-1 Certified Reference Materials. The best results were obtained by sampling blood drops on a decontaminated PVDF filter membrane. After drying under metal-free conditions, the DBSs underwent acidic digestion and were analyzed with ICP MS. The method was then validated for As, Cd, Cu, Pb, Mo, Se and Zn. Using a matrix-matched calibration curve, the recovery levels ranged from 96% to 117%. The repeatability and reproducibility were generally below 15%. Limits of quantification ranging from 0.5 to 50 μg/L. In order to investigate the analytical procedure under real sampling conditions, the results obtained from DBSs and liquid blood aliquots (less subject to contamination) from two adult subjects were compared.     

A rapid,simple and sensitive loop-mediated isothermal amplification method to detect Anaplasma bovis in sheep and goats samples

A loop-mediated isothermal amplification (LAMP) technique has been widely used in detecting the nucleic acid of various pathogenic bacteria. In this study, a set of four LAMP primers was designed to specifically test Anaplasma bovis. The LAMP assay was performed at 62 °C for 60 min in a water bath. The specificity was confirmed by amplifying A. bovis isolate, while no cross reaction was observed with other five pathogens (Anaplasma bovis, Anaplasma phagocytophilum, Theileria luwenshuni, Babesia motasi and Schistosoma japonicum). The sensitivity of LAMP was 5 × 10⁰ copies/μL, 100 times more than that of conventional PCR (5 × 10² copies/μL). Of 120 blood DNA extracted from sheep and goats field samples, 81 (67.5%), 22 (18.3%) and 43 (35.8%) were positively detected by LAMP, conventional PCR and nested PCR, respectively. The findings indicated that the developed LAMP assay is a new convenient tool for rapid and cost-effective detection of A. bovis.     

Development of real-time RT-PCR for detecting viable Cochlodinium polykrikoides (Dinophyceae) cysts in sediment

Morphological observations have confirmed that cysts are produced by dinoflagellates. However, finding a seed bed or unknown cysts in field samples by microscopy is extremely time consuming. Real-time PCR has been used to facilitate the detection of dinoflagellate cysts in sediment. However, DNA from dead vegetative cells remaining on the surface sediment may persist for a long period of time, which can cause false positive DNA detection. In this study, a non-quantitative RNA targeted probe using real-time RT-PCR was developed for detection of viable cysts in sediment. Large-subunit rRNA was used to develop a species-specific RNA targeted probe for the ichthyotoxic dinoflagellate Cochlodinium polykrikoides. The sediment samples were sieved and incubated at 30 °C for 3 h prior to RNA extraction to remove RNA from dead cells remaining in the sediment. Nested-PCR was conducted to maximize assay sensitivity. A field survey to determine the distribution of cysts at 155 sampling stations in the western and southern part of the Korean peninsula showed that C. polykrikoides cysts were detected at five sampling stations.     

Determination of methocarbamol concentration in human plasma by high performance liquid chromatography–tandem mass spectrometry

A simple and reproducible high performance liquid chromatography–tandem mass spectrometric method was developed for methocarbamol analysis in human plasma. Methocarbamol and the internal standard (IS) were extracted by a protein precipitation method. Under isocratic separation condition the chromatographic run time was 3.0 min. The calibration curve was linear over a range of 150–12,000 ng/mL with good intraday assay and interday assay precision (CV% < 10.9%). The method was proven to be sensitive and selective for the analysis of methocarbamol in human plasma for bioequivalence study.     

Activation of nylon net and its application to a biosensor for determination of glucose in human serum

A glucose biosensor using a glucose oxidase (GO_x)-immobilized nylon net with glutaraldehyde as cross-linking reagent and an oxygen (O₂) electrode for the determination of glucose has been fabricated. The detection scheme was based on the utilization of dissolved O₂ in oxidation of glucose by the membrane bound GO_x. Crucial factors including O-alkylation temperature, reaction times of nylon net with dimethyl sulfate, l-lysine, and glutaraldehyde, and enzyme loading were examined to determine the optimal enzyme immobilization conditions for the best sensitivity of the developed glucose biosensor. In addition, the effects of pH and concentration of phosphate buffer on the response of the biosensor were studied. The glucose biosensor had a linear range of 18 μM to 1.10 mM with the detection limit of 9.0 μM (S/N = 3) and response time of 80 s. The biosensor exhibited both good operational stability with over 200 measurements and long-term storage stability. The results from this biosensor compared well with those of a commercial glucose assay kit in analyzing human serum glucose samples.     

Sampling plans for estimating pepper fruit damage levels by Oriental tobacco budworm,Helicoverpa assulta (Guenee), in hot pepper fields

Sequential sampling programs for the management of Oriental tobacco budworm, Helicoverpa assulta (Guenee), on red hot peppers were developed using the data of damaged pepper fruits by H. assulta. Taylor's power law indicated that the damaged pepper fruits were distributed randomly in hot pepper fields. A fixed-precision-level sequential sampling plan for classifying fruit damage density levels at a critical density of 2 damaged fruits per plant was developed to assist in decision making. The sequential classification sampling plan was evaluated using the operating characteristic (OC) and the average sample size (ASN) curves. The OC and ASN curves indicated that this sampling plan was robust and properly classified the population density. A resampling simulation demonstrated that average actual sampling precision value at D = 0.25 was ≤ 0.25. With sequential sampling for classifying the damaged fruit levels in terms of a critical density, sample size was fixed to 18 plants. The fixed-precision-level sequential sampling plan developed in this study should greatly enhance the monitoring efficacy and provide practical solutions suitable for reliable decision-making process in the management of H. assulta.     

Biosorption of Basic Orange using dried A. filiculoides

In order to understand the biosorption of Basic Organic (BO) textile dye on dried Azolla filiculoides (A. filiculoides), batch experiments were conducted under various conditions. The results show that biosorption of BO on dried A. filiculoides was dependent on the initial solution pH, biosorbent dosage, contact time and the initial BO concentration. Using the Langmuir equation, the biosorption capacity (q_m) for BO was 833 mg/g at 303 K. The kinetic study suggested that the mechanism of biosorption was due to ion-exchange physisorption via the intra-particle diffusion and chemisorption on the external surface of dried A. filiculoides. Different techniques were used to characterize dried A. filiculoides and indicated that the biomass had a high cation exchange capacity (93.6 mmol/100 g), a large specific surface area (80.35–422.89 m²/g) and contained various functional groups which may play an important role in the physisorption and chemisorption of BO on the surface of A. filiculoides. The results showed that the removal ratio of BO reached 79.3% from wastewater containing 100 mg/L BO, indicating that the biomass could be used as a potential biosorbent for the removal of BO from industrial wastewater.     

Evaluation of IFN-γ polymorphism +874 T/A in patients with recurrent tonsillitis by PCR Real Time Mismatch Amplification Mutation Assay (MAMA Real Time PCR)

Interferon gamma (IFN-γ) is an important cytokine that plays a crucial role in the balance between normal and pathological immune response. Defect of IFN-γ can give a predisposition to infectious disease, autoimmune pathologies and tumours. Different polymorphisms in this gene have been described, in particular the single nucleotide polymorphism (SNP) + 874 1 T/A that may affect IFN-γ gene expression. Several techniques can be used for the detection of SNPs. In this work two PCR Real Time assays were developed, an Amplification Refractory Mutation System (ARMS) and a Mismatch Amplification Mutation Assay (MAMA). Twenty-seven samples from patients (tonsillectomy) and 85 from donor’s blood bank were considered. As a result, 78/85 controls (91.7%) and 25/27 patients (92.6%) were heterozygosis, considering the ARMS-PCR; 55/85 (64.7%) and 14/27 (51.9%) were heterozygosis using MAMA-PCR assay. Fourteen of 85 (16.5%) and 8/27 (29.6%) were homozygosis A, 16/85 (18.8%) and 5/27 (18.5%) presented homozygosis T, taking into account the MAMA-PCR. There are statistically difference between the two assay with p < 0.0001 at Chi-square test. Our preliminary data suggest that tonsillectomy patients had a statistical trend to possess the low IFN-γ polymorphism when compared with control subject (p = 0.3) but is not statistically significant. In conclusion the Real time MAMA-PCR assay has several advantages over other SNP identification techniques such as rapidity, reliability, easily to perform in one working day and applicable in clinical molecular diagnostic laboratories, although sequencing remains the gold standard.     

Long-term dietary effects on substrate selection and muscle fiber type in very-long-chain acyl-CoA dehydrogenase deficient (VLCAD−/−) mice

Dietary fat restriction and increased carbohydrate intake are part of treatment in very-long-chain acyl-CoA dehydrogenase (VLCAD)-deficiency, the most common defect of long-chain fatty acid oxidation. The long-term impact of these interventions is unknown. We characterized here the effects of a fat-reduced, carbohydrate-enriched diet and an increased fat intake on energy metabolism in a mouse model of VLCAD-deficiency.Wild-type and VLCAD^?/? mice were fed one year either with a normal (5.1%), a high fat (10.6%) or a low-fat, carbohydrate-enriched (2.6%) diet. Dietary effects on genes involved in lipogenesis, energy homeostasis and substrate selection were quantified by real-time-PCR. Acylcarnitines as sign of impaired energy production were determined in dried blood spots and tissues. White skeletal muscle was analyzed for muscle fiber type as well as for glycogen and triglyceride content.Both dietary modifications induced enhanced triacylglyceride accumulation in skeletal muscle and inhibition of glucose oxidation. This was accompanied by an up-regulation of genes coding for oxidative muscle fiber type I and a marked accumulation of acylcarnitines, especially prominent in the heart (164 ± 2.8 in VLCAD^?/? vs. 82.3 ± 2.1 in WT μmol/mg) under a low-fat, carbohydrate-enriched diet.We demonstrate here that both dietary interventions with respect to the fat content of the diet reverse endogenous compensatory mechanisms in muscle that have evolved in VLCAD^?/? mice resulting in pronounced energy deficiency. In particular, the low-fat carbohydrate-enriched diet was not effective in the long term. Further experiments are necessary to define the optimal energy provision for fatty acid oxidation defects.     

Development of an immunochromatographic assay for diagnosing the production of IMP-type metallo-β-lactamases that mediate carbapenem resistance in Pseudomonas

Rapid and reliable detection of carbapenem-resistant bacteria is an important infection-control measure and a crucial aspect of antimicrobial chemotherapy. IMP-type metallo-β-lactamase (MBL) is an emzyme that mediate carbapenem resistance in bacteria. Here, an immunochromatographic assay was newly developed using novel monoclonal antibodies (mAbs) recognizing IMP-type MBL. Epitope mapping of mAbs and mutational analysis of the epitope region in IMP antigen suggested that the mAbs could react to all known subtypes of IMP-type MBL. Evaluation of the assay using Pseudomonas aeruginosa strains (n = 248) showed that the results of the immunochromatographic detection of the IMP-type MBLs were fully consistent with those of the PCR analysis for bla_IMP genes, showing false positives and negatives. All positive strains were resistant to carbapenem (MIC ≥ 16 μg/ml). The assay also accurately distinguished the production of IMP-type MBLs in Pseudomonas putida, Acinetobacter baumannii, and Alcaligenes xylosoxidans. The detection limit of the assay was 5.7 × 10⁴ cfu per test. Taken together, these data suggest that the developed assay can be used for rapid and reliable diagnosis of the production of IMP-type MBLs in Gram-negative bacteria.     

Microbiological assay for quantitative determination of polyoxin B

Polyoxin B, an antifungal agro-antibiotic, is used for treating serious plant diseases. Until now there have been no reports on the antibiotic quantification in liquid in spite of its need for fermentation studies. This work reported a microbiological assay, the cylinder-plate method, for the determination of polyoxin B. The results of assay were treated statistically by analysis of variance (ANOVA) and were found linear in the range of 10–500 μg/ml (r² = 0.998), precise (intra-assay: relative standard deviation (R.S.D.) = 0.64; inter-assay: R.S.D. = 1.70) and accurate. This newly established method was applied to determine the antibiotic titer in Streptomyces cacaoi fermentation with comparison to HPLC analysis. The microbiological assay was satisfactory for polyoxin B quantification, and a simple, sensitive, cost-effective and specific agar diffusion bioassay for polyoxin B was thus developed. This work also demonstrated the usefulness of the microbiological assay for quantitative analysis of antibiotics in fermentation.