Method For Simultaneous Screening Research Articles

High throughput method for simultaneous screening of membrane permeability and toxicity for discovery of new cryoprotective agents.

Vitrification is the most promising method for cryopreservation of complex structures such as organs and tissue constructs. However, this method requires multimolar concentrations of cell-permeant cryoprotective agents (CPAs), which can be toxic at such elevated levels. The selection of CPAs for organ vitrification has been limited to a few chemicals; however, there are numerous chemicals with properties similar to commonly used CPAs. In this study, we developed a high-throughput method that significantly increases the speed of cell membrane permeability measurement, enabling ~100 times faster permeability measurement than previous methods. The method also allows assessment of CPA toxicity using the same 96-well plate. We tested five commonly used CPAs and 22 less common ones at both 4 °C and room temperature, with 23 of them passing the screening process based on their favorable toxicity and permeability properties. Considering its advantages such as high throughput measurement of membrane permeability along with simultaneous toxicity assessment, the presented method holds promise as an effective initial screening tool to identify new CPAs for cryopreservation.

Doping control analysis of myo-inositol trispyrophosphate and 10 bisphosphonates in equine plasma by ion chromatography-mass spectrometry and its application to clodronic acid horse administration.

Bisphosphonates and myo-inositol trispyrophosphate (ITPP) are two classes of difficult-to-detect polar drugs that are prohibited under the rules of racing. ITPP is a drug capable of increasing the amount of oxygen in hypoxic tissues, and studies have shown that administration of ITPP increases the maximal exercise capacity in mice. The properties of ITPP make it an ideal candidate as a doping agent to enhance performance in racehorses. In recent years, ITPP had indeed been detected in racehorses and confiscated items. As for bisphosphonates, it is especially critical to control their use as since February 2019, the International Agreement on Breeding, Racing and Wagering (IABRW) by the International Federation of Horseracing Authorities (IFHA) had identified specific conditions on which bisphosphonates should not be administered to a racehorse. A recent review of literature shows that there is yet a simultaneous screening method for detecting ITPP and bisphosphonates in equine samples. This paper describes an efficient ion chromatography high-resolution mass spectrometry (IC-HRMS) method for the simultaneous detection of ITPP and 10 bisphosphonates at sub-parts-per-billion (ppb) to low-ppb levels in equine plasma after solid-phase extraction (SPE) and its application to an administration study of clodronic acid in horses.

Development of a method for simultaneous screening of four natural-derived steroids and their analogues used as dietary supplements via liquid chromatography-quadrupole-time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry

Natural-derived steroids and their analogues are present in various plants and insects. To minimize the chance of missing a positive doping test and avoiding potentially serious health problems, adequate screening methods are necessary for the detection of a wide range of natural-derived steroids and their analogues in dietary supplements. In this study, an accurate and simple liquid-chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to determine and quantify the natural-derived steroids and their analogues according to the International Conference on Harmonization of technical Requirements for Registration of Pharmaceuticals for Human Use guidelines. The validation results indicating excellent extraction efficiency and low matrix effects show that the LC-MS/MS method is reliable for the detection of natural-derived steroids and their analogues. In addition, we established the ion fragmentation of turkesterone and ion fragmentation of four natural-derived steroids and their analogues. The validated method was applied to 60 dietary supplements purchased online and in person from international vendors in 2020. Ecdysterone and 5α-hydroxylaxogenin were detected respectively in 3 and 14 of 60 dietary supplements. Especially, a high amount of 5α-hydroxylaxogenin, an FDA-unapproved ingredient, was detected in two of dietary supplements (44.4 and 32.3 mg/g). This component should be controlled since it may cause unexpected side effects if administered excessively. Thus, this method will be helpful for the continuous control and supervision of unlicensed dietary supplements containing natural-derived steroids and their analogues.

Establishment of modified QuEChERS-GC–MS-LC–MS/MS method for simultaneous screening of multi-class multi-pesticide residues in betelvine and consumer risk assessment

Betelvine (Piper betel L.) in India is grown as an important cash crop with great demand in several countries. Due to its raw intake, the presence of undesirable pesticides in betelvine is an alarming issue of global concern. Therefore, a reliable method is needed to monitor their residues. In this study, a modified QuEChERS method was developed and validated to screen fifty-seven (57) widely used multi-class pesticide residues using gas chromatography–mass spectrometry (GC–MS) and liquid chromatography with tandem mass spectrometry (LC-MS/MS). The sample was extracted with acetonitrile and cleaned in dispersive solid-phase extraction (dSPE) with graphitized carbon black (GCB) + primary secondary amine (PSA) + Florosil, PSA + C18, and C18 + GCB sorbent combinations to assess matrix interference cleanup efficiencies. The optimized method produced remarkable linearity from 0.01 to 1.0 mgL-1 with satisfactory limits of quantification from 0.01 to 0.05 mgL-1. The best-spiked recovery of target analytes in the GCB + PSA + Florosil combination was 70–120% with precision RPDs ≤ 17.97%, demonstrating good adsorption ability for co-extractives. Matrix effect (ME) values ranged between −19.74% and + 23.6%. Collected market samples (75) from the designated districts of West Bengal, India were screened and 9% were found to be contaminated with some non-recommended pesticides like carbendazim and tebuconazole. Human risk indexes (HRI) of the determined xenobiotics were also estimated (0.007 to 0.62), which were fortunately within the acceptable limit (HRI ≤ 1.0). So, the developed modified QuEChERS method might be beneficial for quick and accurate screening of multi-class multi-pesticide residues in betelvine intended for export and food safety.

Application of a simultaneous screening method for the detection of new psychoactive substances in various matrix samples using liquid chromatography/electrospray ionization tandem mass spectrometry and liquid chromatography/quadrupole time‐of‐flight mass spectrometry

Recently, new psychoactive substances (NPS) emerged as a public health risk. Particularly, their chemical structures are modified to avoid detection. Synthetic NPS with effects similar to those of illegal drugs have been recently detected and synthesised worldwide, including MDMB-FUBINACA and APINAC, making it essential to rapidly and accurately detect NPS. Fourteen NPS with similar structures were selected and their structures identified using 1 H and 13 C NMR spectroscopy. Additionally, we proposed the fragmentation pattern of each compound using LC-QTOF-MS. A simultaneous analytical method using ESI-LC-MS/MS was also developed and applied to real samples to detect 14 NPS. The method was validated based on the specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, matrix effect, and stability according to international validation guidelines. The established method was used to screen 65 different matrix samples using LC-ESI-MS/MS. By comparing the calculated product ion ratios with those of standards, 2C-B in one real samples and 5F-MDMB-PICA in 20 samples were identified. For re-confirmation of detected compounds, each fragmentation pattern of each compound was compared to that of each standard using LC-QTOF-MS. In this study, LC-QTOF-MS were used to elucidate the structures and fragmentation patterns of 14 NPS. A simultaneous method was developed using LC-ESI-MS/MS, which was applied to 65 real samples. The presented method and results can assist in ensuring the safety of public health from illegal adulteration.

A Rapid Thin Layer Chromatographic Method for Simultaneous Screening of Albendazole and Ivermectin in Formulations Prescribed for Human or Veterinary Use

An easy and selective thin layer chromatographic method has been developed and experimentally validated for the simultaneous screening of most commonly used anthelmintic drugs i.e. albendazole and ivermectin. Separation of these compounds was attained on silica gel 60 F254 pre-coated thin layer chromatographic plate using an optimized mobile phase of diisopropyl ether:ethyl acetate:glacial acetic acid in the ratio of 7:3:0.1 (v/v), respectively at pH 3.5. The calculated Rf values for albendazole and ivermectin were 0.65 and 0.38, respectively and the LOD was found to be 25 μg/ml and 30 μg/ml for albendazole and ivermectin, respectively. The developed method is selective, sensitive, robust, cost effective, eco-friendly, rapid as well as easy to perform. The developed method was successfully applied for the analysis of albendazole and ivermectin in pharmaceutical preparations marketed as oral suspensions, powder, tablets and injectable of the single or combined dosage forms for human as well as veterinary use. It could also be applied for the simultaneous analysis of both the compounds in other samples.

Non-targeted screening and determination of steviol glycosides in Chinese liquor and beverages by ultra high performance liquid chromatography-quadrupole/Orbitrap mass spectrometry

A method for simultaneous screening and determination of steviol glycosides in Chinese liquor and beverages by hydrophilic interaction ultra-high performance liquid chromatography-quadrupole/Orbitrap high resolution mass spectrometry (HILIC-UHPLC-Q/Orbitrap HRMS) was developed. In addition, the MS fragment path ways of steviol glycosides were characterized. The sample was diluted and filtrated before analysis, separated by a Waters Xbridge Amide column (150 mm×4.6 mm, 3.5 μm), and determined via UHPLC-Q/Orbitrap HRMS. The analytes were electronic using electro spray ion source in the negative mode, and the data were collected using full mass scan and data-dependent MS2 scan. Good linear relationships (10-1000 μg/L) were obtained. The limits of detection ranged from 0.3 to 20 μg/L. The mean recoveries varied from 81.9% to 106% at the spiked levels of 10-100 μg/kg, and the relative standard deviations ranged from 0.1% to 9.3% (n=3). Two characteristic fragment ions of steviol glycosides were identified by the analysis of fragment ion spectra of eight types of steviol glycosides standards. Non-targeted screening of unknown steviol glycosides in samples was carried out on the basis of the identified characteristic fragment ions. The proposed method is comprehensive, accurate, and sensitive for rapid screening and determination of sweeteners in food samples.

Rapid screening and quantification of multi-class antibiotic pollutants in water using a planar waveguide immunosensor.

Antibiotics are commonly used in livestock-related agriculture and aquaculture, but they also remain in water and potentially threaten human health. Immunosensors are attractive tools for the rapid detection of antibiotics in water due to their high sensitivity and low costs. However, the simultaneous detection of multi-class antibiotics remains a challenge due to the limited number of detection sites on the immunochip. Also, matrix effects hinder the practical application of these sensors. This paper presents a method for multi-class antibiotic detection in real water using a planar waveguide immunosensor (PWI). We integrate the screening and quantitive detection sites on the same immunochip, and a single screening detection site could detect multi-class antibiotics from the same family, increasing the detection types of analytes. In addition, to eliminate the matrix effects, we develop a testing buffer for real water detection, so that complex pretreatments of the samples can be omitted. Using our sensor and testing buffer, we detect 14 different antibiotics in real water. Lincomycin can be detected with a detection limit of 0.01 μg L−1, and 13 quinolones can be screened in a single assay. These results demonstrate that this planar waveguide immunosensor is capable of simultaneous screening and quantification of multi-class antibiotic pollutants and is expected to be applied for practical environmental monitoring.

Sensitive Liquid Chromatography/Tandem Mass Spectrometry Method for the Simultaneous Determination of Risperidone, Olanzapine, Quetiapine, Clozapine, Ziprasidone, Perospirone, Aripiprazole and Blonanserin in Human Serum

The sensitive method for the simultaneous determination of eight atypical antipsychotic drugs (risperidone, olanzapine, quetiapine, clozapine, ziprasidone, perospirone, aripiprazole and blonanserin) in human serum has been developed based on a high-performance liquid chromatography-tandem mass spectrometry (LC/MS/MS) with electrospray ionization (ESI). The chromatographic separation was performed on a Mightysil-RP-18 MS column (2.0 mm × 150 mm, particle size 3 μm). The mobile phase consisted of 10 mM formic ammonium buffer (pH 6.0) and acetonitrile and was delivered at a flow rate of 0.20 mL/min. The triple quadrupole mass spectrometer was operated in positive ion mode, and multiple reaction monitoring was used for drug quantification. Solid-phase extraction of the eight antipsychotic drugs added to the human serum was performed with an Oasis®HLB extraction cartridges column. The method was linear for the investigated drugs over the concentration range of 20 - 100 ng/mL. The recoveries of these drugs were in the range of 81.3% - 140%. The intraday and inter day standard deviation (SD) values for all analytes were <0.10. Therefore, the selectivity, accuracy and precision of this method are sufficient for clinical and forensic studies. This simultaneous screening method will be also applicable to analyze other atypical antipsychotic drugs.

Analysis of new psychoactive substances in oral fluids by means of microextraction by packed sorbent followed by ultra‐high‐performance liquid chromatography–tandem mass spectrometry

In recent years, new drugs, commonly known as new psychoactive substances (NPS), appeared on the market, which include, among others, synthetic cannabinoids, cathinones, and tryptamine analogs of psilocin. The aim of this work was to develop and validate a new method for simultaneous screening and quantification of 31 NPS in oral fluid by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The chosen target analytes represented different chemical and toxicological NPS classes, such as synthetic cathinones, piperazines, phenethylamines, synthetic cannabinoids, and their metabolites. The procedure involved a rapid sample preparation based on protein precipitation followed by clean-up utilizing microextraction by packed sorbent (MEPS); the quantitative analysis was performed by UHPLC-MS/MS. The MEPS clean-up, regardless of non-quantitative recoveries for some analytes, provided an effective removal of interfering compounds, as demonstrated by reduced matrix effects found at different concentrations for all the analytes. The validation protocol, based on SWGTOX guidelines, demonstrated the suitability of the proposed method for quantitative analysis: linearity range ranged over 3 or 4 orders of magnitude; precision and accuracy tests gave RSD% values below 25%, and accuracy ranged from 85.9% to 107%, accomplishing SWGTOX requirements. Limits of detection (LODs) ranged between 0.005 ng/mL and 0.850 ng/mL and limits of quantification (LOQs) from 0.015 to 2.600 ng/mL.

Assessment of simpler calibration models in the development and validation of a fast postmortem multi-analyte LC-QTOF quantitation method in whole blood with simultaneous screening capabilities using SWATH acquisition.

In postmortem toxicology, fast methods can provide a triage to avoid unnecessary autopsies. Usually, this requires multiple qualitative and quantitative analytical methods. The aim of the present study was to develop a postmortem LC-QTOF method for simultaneous screening and quantitation using easy sample preparation and reduced alternative calibration models. Hence, a method for 24 highly relevant substances in forensic toxicology was fully validated using the following calibration models: one-point external, one-point internal via corresponding deuterated standards, multi-point external daily calibration, and multi-point external weekly calibration. Two hundred microliters of postmortem blood were spiked with internal deuterated standard mixture and extracted by acetonitrile protein precipitation. Analysis was performed on a Sciex 6600 QTOF instrument with ESI+ mode using data-independent acquisition (DIA) namely sequential window acquisition of all theoretical mass spectra (SWATH). Validation of the different calibration models included selectivity, autosampler stability, recovery, matrix effects, accuracy, and precision for 24 substances. In addition, corresponding deuterated analogs of 52 substances were included to the internal standard mix for semi-quantitative concentration assessment. The simple protein precipitation provided recoveries higher than 55 and 75% for all analytes at low and high concentrations, respectively. Accuracy and precision criteria (bias and imprecision ± 15 and ± 20% near the limit of quantitation) were fulfilled by the different calibration models for most analytes. The validated method was successfully applied to more than 100 authentic postmortem samples and 3 proficiency tests. Furthermore, the one-point internal calibration via corresponding deuterated standard proved to be a considerably time saving technique for 76 analytes. Graphical abstract One-point and multi-point calibration and the resulting beta-tolerance intervals from method validation.

Online turbulent flow extraction coupled with liquid chromatography–tandem mass spectrometry for high throughput screening of anabolic steroids in horse urine

A high throughput method for simultaneous screening of anabolic steroids and their metabolites (4-esterendione, trenbolone, boldenone, oxandrolone, nandrolone, methandrostenolone, testosterone, 1-androstendione, ethisterone, normethandrolone, methyltestosterone, 16β-Hydroxystanozolol, epitestosterone, bolasterone, norethandrolone, danazol, stanozolol and androstadienone) in equine urine by online turbulent flow extraction coupled with liquid chromatography-tandem mass spectrometry was developed. The use of turbulent flow chromatography could simplify pretreatment of horse urine, which has complex matrices as well as high viscosity. The urine was extracted by mixed-mode cation exchange solid phase extraction, and hydrolyzed using β-glucuronidase/arylsulfatase. Then, the sample was automatically loaded on the TurboFlow Cyclone extraction column for removal of further matrix, followed by separation on a fused core C18 column before MS/MS detection. Optimization and validation of the method were discussed in detail. All analytes were rapidly detected within 10min with high sensitivity (picogram to nanogram per milliliter level), and no interference was observed. The linearity range was from 0.1–10ng/mL for nine steroids and 1.0–50ng/mL for the others, with correlation of coefficient values over 0.995. Precision and accuracy ranged from 0.1 to 14.5% and 1.7 to 12.4%, respectively. The developed method was successfully applied to the analysis of anabolic steroids in horse urine after administration of a model drug.

Validated LC-MS-MS Method for Simultaneous Analysis of 17 Barbiturates in Horse Plasma for Doping Control.

A rapid and sensitive method for simultaneous screening, quantification and confirmation of 17 barbiturates in horse plasma using liquid chromatography-tandem mass spectrometry is described. Analytes were recovered from plasma by liquid-liquid extraction using methyl tert-butyl ether, separated on a C18 column, and analyzed in negative electrospray ionization mode. Multiple-reaction monitoring was employed for screening and quantification. Confirmation for the presence of the analytes was achieved by comparing ion intensity ratio. The ranges for limits of detection, quantification and confirmation were 0.003-1 ng/mL (S/N ≥ 3), 0.01-2.5 ng/mL and 0.02-5 ng/mL, respectively. The linear dynamic range of the method was 0.1-100 ng/mL. The precision and accuracy at 0.5, 5 and 50 ng/mL of all 17 barbiturates during intra-day assay were 1.6-8.6% and 96-106%, respectively. For inter-day assay, precision and accuracy at the same three concentrations were 2.6-8.9% and 96-106%, respectively. Analysis of all 17 analytes was completed within 7 min. Thus, the present method is fast, simple, sensitive and reproducibly reliable.

A Rapid and Sensitive Method for Simultaneous Screening of Nine Foodborne Pathogens Using High-Performance Liquid Chromatography Assay

We developed a multiplex PCR-based procedure followed by high-performance liquid chromatography (mPCR-HPLC) assay for high-throughput screening foodborne pathogens, including Salmonella spp., Listeria monocytogenes, Enterobacter sakazakii, Staphylococcus aureus, Shigella spp., Escherichia coli O157:H7, Vibrio parahaemolyticus, Vibrio cholerae, and Vibrio vulnificus. Vibrio species-specific primers were designed targeting dnaJ gene, and pathogen-specific primers for L. monocytogenes, Salmonella spp., E. sakazakii, E. coli O157:H7, Shigella spp., and S. aureus were designed targeting iap, fimY, 16S ribosomal RNA (rRNA), rfbE, ipaH, and 442 genes, respectively. PCR products were analyzed using WAVE-4500 DNA system equipped with PS-DVB-C18 particles DNASep column and each specific amplicon generated a characteristic chromatographic profile. Detection limit of mPCR-HPLC assay was ca. 101 CFU/mL in pure cultures and less than 102 CFU/g in contaminated matrixes. A total of 395 bacterial strains were used for specificity testing of the mPCR-HPLC assay, and specific HPLC profiles were only produced in strains belonging to the target, showing a high specificity. Applying the assay to 2677 samples collected from clinical, food, and environmental sources revealed that 917 samples were positive, in accordance with bacterial isolation. The high sensitivity and specificity of mPCR-HPLC assay indicate its great potential to be a powerful tool for high-throughput screening foodborne pathogens.

"One-shot" analysis of PDE-5 inhibitors and analogues in counterfeit herbal natural products using an LC-DAD-QTOF system.

A highly selective and robust method for simultaneous screening and confirmation of target and non-target phosphodiesterase type 5 (PDE-5) inhibitor analogues within a single chromatographic run in counterfeit herbal products was developed. The protocol, based on an easy and rapid extraction with a water/acetonitrile 1% formic acid solution, followed by sonication and centrifugation, exploits an LC-diode array detector-quadrupole-time-of-flight (DAD-QTOF) system. The extraction method was optimized both at high concentrations and at trace levels. These two situations are typically encountered in pharmaceutical formulations and herbal food supplements. Carryover effects, never reported before and occurring mainly for vardenafil, were overcome using a polymer-based column. An in-house validation was carried out using five blanks of different bulk matrices spiked with seven standard analytes, namely yohimbine, sildenafil, vardenafil, tadalafil, homosildenafil, pseudovardenafil and hydroxyhomovardenafil. Reliable quantitation was possible using a conventional standard solution for all the pharmaceutical and herbal samples considered, as matrix effects were eliminated. Accuracy ranged from 80.9 to 108.1% with overall relative standard deviation (RSD) <11% (N = 15), measured at 1.0, 5.0 and 10.0μg/g. Limits of detection (LODs) obtained ensured the determination of cross contaminations at nanogram per gram levels. A database with 82 PDE-5 inhibitor analogues was implemented for automatic non-target analysis. Among the 26 samples of dietary supplements and herbal remedies bulk marketed for erectile dysfunctions, three samples were found to be contaminated with both registered and unregistered synthetic PDE-5 inhibitors, i.e. yohimbine, sildenafil, dimethylsildenafil and thiodimethylsildenafil or thiomethisosildenafil. The occurrence of such contaminations, both at trace levels and at pharmaceutical dosage, indicates the illicit use of synthetic PDE-5 analogues. Graphical Abstract Examples of pharmaceutical formulations and herbal natural products marketed for the erectile dysfunction.

An analysis method for simultaneous screening of deoxyribonucleic acid-binding active compounds and investigating their mechanisms by ultra-fast liquid chromatography tandem mass spectrometry coupled with fluorescence detection technology

DNA has been known as the cellular target for many cytotoxic anticancer agents over the years. Discovering DNA-binding compounds has become an active research area, while various DNA-binding mechanisms make the drug discovery even more difficult. In this article, we present a novel analysis method to rapidly identify specific DNA-binding compounds from Pyrrosia lingua (Thunb.) using DNA-dual-fluorescent probes, ethidium bromide and Hoechst 33258, with the technology of ultra-fast liquid chromatography-diode array detector-tandem mass spectrometry and dual-wavelength fluorescence detector (UFLC-DAD-MSn-DFLD). Sixty-two compounds were identified, of which 22 were found to be active in DNA-binding. After investigation of their dose-response behaviors and structure–activity relationships, chlorogenic acids and flavonoid glycosides were found to be DNA-binders via both minor groove-binding and intercalation modes. The precision, reproducibility and stability of this method were validated by vitexin. The established system was sensitive, precise, and reliable to be used for both screening of DNA-binding compounds and investigating of their mechanisms.

Simultaneous Screening of 177 Drugs of Abuse in Urine Using Ultra-performance Liquid Chromatography with Tandem Mass Spectrometry in Drug-intoxicated Patients

ObjectiveThe demand for rapid and broad clinical toxicology screening methods to identify drugs of abuse and medicinal drugs is increasing steadily. Liquid chromatography-tandem mass spectrometry (LC-TMS) is increasingly used to screen for drugs of abuse and to identify a wide range of drugs and metabolites in clinical samples. We revised a high-throughput and rapid ultra-performance (UP) LC-TMS method for simultaneous screening of 177 of the most prevalent medicinal drugs and drugs of abuse in urine and validated the quality of performance using system suitability mixture (SSM) and quality control (QC) materials.MethodsWe assessed the limits of detection (LOD) using high concentrations of the test substances. The method was applied to 473 urine samples obtained from patients intoxicated with drugs who visited the emergency center.ResultsThe retention time, peak area, and total ion chromatogram of the SSM and QC materials were within the acceptance criteria of the pre-defined acceptance interval. The LODs were <62 ng/ml for 12 commonly encountered drugs. In total, 418 patients (88.4%) tested positive for one or more medicinal drugs or drugs of abuse. Twenty-eight drugs were detected over ten times; the most commonly detected were zolpidem, ephedrine, paracetamol, and chlorpheniramine.ConclusionThe UPLC-TMS method provided excellent performance for simultaneous screening of a large number of the drugs of abuse in urine samples. We conclude that this robust technique is useful for screening for a large number of drugs and for rapid screening of the most commonly encountered substances in emergency cases.

Multiresidue Detection of Fluoroquinolones: Specificity Engineering of a Recombinant Antibody with Oligonucleotide-Directed Mutagenesis

Screening of a group of antibiotics from foodstuffs has traditionally relied on sophisticated chemical or physical analysis methods, such as liquid chromatography and mass spectrometric applications. The equipment for these techniques is expensive and not always applicable for high throughput screening. There is a need for an easy and cost efficient detection method for simultaneous screening of structurally similar compounds. Here we describe the engineering of a recombinant antibody which was subjected to oligonucleotide targeted random mutagenesis to emphasize the generic specificity of fluoroquinolone binding. Phage display together with small sized fluoroquinolone derivatives was used to find antibodies of high affinity and generic specificity. The most improved antibody was used to develop a time-resolved fluorescence immunoassay which was further optimized and applied for the detection of fluoroquinolone residues from spiked whole milk samples. The assay can be used to efficiently screen all European Agency for the Evaluation of Medicinal Products (EMEA) controlled fluoroquinolones from whole milk samples with detection levels ranging from 0.2 to 68 μg L(-1).

A Rapid Method for Simultaneous Screening of Multi-Gene Mutations Associated with Hearing Loss in the Korean Population

Hearing loss (HL) is a congenital disease with a high prevalence, and patients with hearing loss need early diagnosis for treatment and prevention. The GJB2, MT-RNR1, and SLC26A4 genes have been reported as common causative genes of hearing loss in the Korean population and some mutations of these genes are the most common mutations associated with hearing loss. Accordingly, we developed a method for the simultaneous detection of seven mutations (c.235delC of GJB2, c.439A>G, c.919-2A>G, c.1149+3A>G, c.1229C>T, c.2168A>G of SLC26A4, and m.1555A>G of the MT-RNR1 gene) using multiplex SNaPshot minisequencing to enable rapid diagnosis of hereditary hearing loss. This method was confirmed in patients with hearing loss and used for genetic diagnosis of controls with normal hearing and neonates. We found that 4.06% of individuals with normal hearing and 4.32% of neonates were heterozygous carriers. In addition, we detected that an individual is heterozygous for two different mutations of GJB2 and SLC26A4 gene, respectively and one normal hearing showing the heteroplasmy of m.1555A>G. These genotypes corresponded to those determined by direct sequencing. Overall, we successfully developed a robust and cost-effective diagnosis method that detects common causative mutations of hearing loss in the Korean population. This method will be possible to detect up to 40% causative mutations associated with prelingual HL in the Korean population and serve as a useful genetic technique for diagnosis of hearing loss for patients, carriers, neonates, and fetuses.

コンベンショナルPCR を用いた &lt;i&gt;Bordetella&lt;/i&gt; 属菌の一斉スクリーニング法

A simultaneous screening method using conventional PCR was developed for the detection and discrimination of Bordetella pertussis, Bordetella parapertussis, and Bordetella holmesii. A formulated multiprex method employing 4 kinds of paired primers on amplification of 4 corresponding different insertion sequences (IS481, IS1001, IS1002 and hIS1001) enabled rapid screening and identification. The detection limits of each DNA extracted from 3 kinds of Bordetella species were 5fg/microL for each. Obscure existences of B. pertussis and B. holmesii at low levels were confirmed with the LAMP method. This multiplex assay was applied to the clinical specimens obtained from patients with pertussis-like symptoms at sentinel clinics under the epidemiological surveillance of infectious diseases of Hyogo prefecture in FY2012. Among 42 nasopharyngeal swabs, B. pertussis was detected from 12 samples including 8 samples collected at outbreak in nursery school. The use of this method for the surveillance of infectious agents enabled us to search for 3 kinds of Bordetella species at once with low costs.