高碑店污水系统病毒污染情况的检测

本文报道用石英粉吸附——洗脱法浓集污水病毒,其回收率为51～90％。对1985年3～7月采集的不同水样进行病毒分离和蚀斑滴定,结果表明,高碑店污水系统中的病毒大部分为肠道病毒。春季以脊髓灰质炎病毒为主,夏季以柯赛奇B3及非肠道病毒为主。不同类型污水中的病毒浓度依次为:原污水58PFU/1;二级污水<19,约为9PFU/1;医用污水<16,约为1PFU/1,地下水<1.7PFU/1,未检出病毒。夏季水样内病毒浓度较春季低。     

Genogroup distribution of F-specific coliphages in wastewater and river water in the Kofu basin in Japan

Aims: To determine the genogroup distribution of F‐specific coliphages in aquatic environments using the plaque isolation procedure combined with genogroup‐specific real‐time PCR. Methods and Results: Thirty water samples were collected from a wastewater treatment plant and a river in the Kofu basin in Japan on fine weather days. F‐specific coliphages were detected in all tested samples, 187 (82%) of 227 phage plaques isolated were classified into one of the 4 F‐specific RNA (F‐RNA) coliphage genogroups and 24 (11%) plaques were F‐specific DNA coliphages. Human genogroups II and III F‐RNA coliphages were more abundant in raw sewage than animal genogroups I and IV, excluding one sample that was suspected to be heavily contaminated with sporadic heavy animal faeces. The secondary‐treated sewage samples were highly contaminated with genogroup I F‐RNA coliphages, probably because of different behaviours among the coliphage genogroups during wastewater treatment. The river water samples were expected to be mainly contaminated with human faeces, independent of rainfall effects. Conclusions: A wide range of F‐specific coliphage genogroups were successfully identified in wastewater and river water samples. Significance and Impact of the Study: Our results clearly show the usefulness of the genogroup‐specific real‐time PCR for determining the genogroups of F‐specific coliphages present in aquatic environments.     

Liquid chromatography-tandem mass spectrometric quantitative determination of the HIV protease inhibitor atazanavir (BMS-232632) in human peripheral blood mononuclear cells (PBMC): practical approaches to PBMC preparation and PBMC assay design for high-throughput analysis

A selective, accurate, and reproducible LC/MS/MS assay was developed and validated for the determination of the HIV protease inhibitor atazanavir (BMS-232632) in human peripheral blood mononuclear cells (PBMC) samples. In addition to the details of the validated LC/MS/MS method, a practical procedure is described in great detail for the preparation of large supplies of control (blank) PBMC from units of blood (each unit of blood is about 500 ml) for making the calibration standards and quality control (QC) samples. The PBMC assay design, intended for high-throughput sample analysis, is also described in some detail in regards to the composition and concentration expressions of the calibration standards and QC samples, the lysing procedure of the PBMC samples, and the final analysis/quantitation procedure. The method involved automated solid-phase extraction (SPE) of atazanavir and a stable isotope analog internal standard (I.S.) using 3M Empore C2-SD 96-well plates. A portion of the reconstituted sample residue was injected onto a YMC Basic analytical column which was connected to a triple quad mass spectrometer for analyte determination by positive-ion electrospray in the selected reaction monitoring (SRM) mode. The standard curve, which ranged from 5 to 2500 fmol per one million cells (fmol/10(6) cells), was fitted to a quadratic regression model weighted by 1/concentration. The lower limit of quantitation (LLOQ) was 5 fmol/10(6) cells. The inter- and intra-run coefficients of variation (CV) for the assay were <9% and the accuracy was 94-104%. Atazanavir was stable in PBMC for at least 24h at room temperature and for at least 129 days at -15 degrees C.     

Use of online-dual-column extraction in conjunction with chiral liquid chromatography tandem mass spectrometry for determination of terbutaline enantiomers in human plasma

An online sample extraction chiral bioanalytical method was developed and validated for the quantification of terbutaline, a beta2-selective adrenoceptor agonist, spiked into human plasma by using two extraction columns and a chiral stationary phase (CSP) in conjunction with liquid chromatography tandem mass spectrometry (LC-MS/MS). In this method, two Oasis HLB extraction columns were used in parallel for plasma sample purification and a Chirobiotic T CSP was used for enantiomeric separation. Atmospheric pressure chemical ionization MS/MS was employed in multiple reaction monitoring mode for the detection and quantification. Subsequent to the addition of an internal standard solution, the plasma samples were directly injected onto the system for extraction and analysis. This method allowed the use of one of the extraction columns for purification while the other was being equilibrated. Hence, the time required for reconditioning the extraction columns did not contribute to the total analysis time per sample, which resulted in a shorter run time and higher throughput. A lower limit of quantification of 1.0 ng/mL was achieved using only 50 microliter of human plasma. The method was validated with a dynamic range of 1.0-200 ng/mL. The intra- and interday precision was no more than 11% CV and the assay accuracy was between 94-106%.     

Quantitation of five nevirapine oxidative metabolites in human plasma using liquid chromatography-tandem mass spectrometry

A multiple-reaction-monitoring LC/MS/MS method for the analysis of nevirapine oxidative metabolites, 2-hydroxynevirapine, 3-hydroxynevirapine, 8-hydroxynevirapine, 12-hydroxynevirapine, and 4-carboxynevirapine, in human plasma was developed and validated. The metabolites were isolated from 50 microL heparinized plasma by enzymatic hydrolysis of the glucuronide conjugates to the free metabolite followed by protein precipitation with acetonitrile. Peaks were quantitated at 3.03 min for the 4-carboxynevirapine metabolite, at 3.72, 4.27, 5.27, and 5.73 min for the positional 2-hydroxynevirapine, 12-hydroxynevirapine, 3-hydroxynevirapine, and 8-hydroxynevirapine metabolites, respectively, and 2.30 min for the internal standard, pirenzepine. The assay was accurate and precise based on assay validation controls over the nominal range of 0.010-1.0 mg/L. The average accuracy at the lowest concentration quality control (QC) sample was 16% (difference from theoretical value) for 8-hydroxynevirapine, all others were closer to their known respective standards. Within- and between-day precisions were within 12% for quality control samples for all five metabolites. Repetitive thawing and freezing did not have an effect on any metabolite through a minimum of three cycles. Thawed samples, remaining in plasma for 4 h before extraction, were within 5% of theoretical value. Stability of the extracted samples on the autosampler at room temperature was evaluated for 48 h and was observed to be within 12% of a fresh analytical sample for 2-hydroxynevirapine and 3-hydroxynevirapine; other metabolites were within 6% of theoretical value. The utility of the analytical method was demonstrated using trough steady-state plasma samples collected from 48 patients in a hepatic impairment study.     

Quantitative determination of tilmicosin in canine serum by high performance liquid chromatography-tandem mass spectrometry

A highly sensitive and quantitative LC/MS/MS assay for the determination of tilmicosin in serum has been developed and validated. For sample preparation, 0.2 mL of canine serum was extracted with 3 mL of methyl tert-butyl ether. The organic layer was transferred to a new vessel and dried under nitrogen. The sample was then reconstituted for analysis by high performance liquid chromatography-tandem mass spectrometry. A Phenomenex Luna C8(2) analytical column was used for the chromatographic separation. The eluent was subsequently introduced to the mass spectrometer by electrospray ionization. A single range was validated for 50-5000 ng/mL for support of toxicokinetic studies. The inter-day relative error (inaccuracy) for the LLOQ samples ranged from -5.5% to 0.3%. The inter-day relative standard deviations (imprecision) at the respective LLOQ levels were < or =10.1%.     

Developing a robust ultrafiltration-LC-MS/MS method for quantitative analysis of unbound vadimezan (ASA404) in human plasma

Ultrafiltration of human plasma in combination with LC-MS/MS has been increasingly used in the quantitative analysis of the free fraction of drug candidates for PK/efficacy assessment. In addition to controlling the pre-incubation and centrifugation temperatures, some important factors that must be investigated and addressed include: (1) possible nonspecific binding, (2) possible impact of freeze/thaw cycles of plasma samples and extended storage of plasma samples at room temperature on the analyte recovery prior to ultrafiltration, and (3) identification of the appropriate assay dynamic range to avoid unnecessary dilutions. These factors were explored in the development and validation of a robust LC-MS/MS assay for the quantitative analysis of unbound vadimezan (ASA404) in human plasma. First, to mimic human physiological conditions, all plasma samples were incubated at ~37°C for a minimum of 30 min after thawing and prior to centrifugation to obtain the ultrafiltrate. Second, by passing the calibration standards and QC samples in plasma ultrafiltrate through the ultrafiltration membrane, the observed non-specific binding of the analyte due to the membrane was corrected. Third, the effects of multiple freeze/thaw cycles and/or storage at room temperature for various periods (4, 8, 16 and 24h) were evaluated to determine the impact on analyte concentrations in the ultrafiltrate from the plasma QC samples. Fourth, the appropriate dynamic range was established to accommodate the expected incurred sample free analyte concentrations. The validated assay has a dynamic range of 30.0-30,000 ng/ml for ASA404 in human plasma ultrafiltrate using a sample volume of 30 μl. Quality control pools containing the analyte were prepared at concentrations of 30.0-22,500 ng/ml to cover the assay calibration range. The intra-assay and inter-assay precision and accuracy were ≤ 15% (CV) and within ± 15% (bias) of the nominal values, respectively, for all measured QC concentrations, including the LLOQ. Freeze/thaw for up to three cycles of the plasma samples and/or the extended human plasma sample exposure to room temperature for up to 24h were confirmed to have no impact on the assay results for the free analyte. The validated method was successfully implemented to support clinical studies for the compound.     

Determination of risperidone and enantiomers of 9-hydroxyrisperidone in plasma by LC-MS/MS

A robust and validated liquid-liquid extraction LC-MS/MS method was developed for population pharmacokinetic analysis and therapeutic drug monitoring of risperidone and the enantiomers of its major active metabolite (+)-and (-)9-hydroxyrisperidone in pediatric patients. The method was rapid, sensitive and used a low sample amount (200 microL), which is very desirable for the pediatric population. The assay was validated from 0.2 to 50 ng/mL in plasma for all analytes. LLOQ for all analytes was 0.2 ng/mL. The extracts were analyzed by normal phase LC-MS/MS. The sample run time was 8 min. Intra- and interday precision for all analytes was < or =6%; method accuracy was between 89 and 99%. Additional experiments were performed to analyze matrix effects and identify a proper internal standard for each analyte. The validated method was used to study risperidone and its enantiomer metabolites in plasma as part of a population pharmacokinetic study in pediatric patients with pervasive developmental disorder (PDD).     

Fortified Sera and Their Use in Environmental Virology

Four commercially available fortified sera were compared to fetal bovine serum (FBS) with regard to their ability to maintain or increase the sensitivity of the Buffalo green monkey (BGM) kidney cell line to viral infection. Nine virus strains and five wastewater samples were used. Fortified sera were comparable to FBS for the enumeration of some viruses by the plaque method and for the detection of virus in wastewater by the most-probable-number assay.     

Fortified sera and their use in environmental virology

Four commercially available fortified sera were compared to fetal bovine serum (FBS) with regard to their ability to maintain or increase the sensitivity of the Buffalo green monkey (BGM) kidney cell line to viral infection. Nine virus strains and five wastewater samples were used. Fortified sera were comparable to FBS for the enumeration of some viruses by the plaque method and for the detection of virus in wastewater by the most-probable-number assay.     

On-chip microextraction for proteomic sample preparation of in-gel digests

Despite the high sensitivity and relatively high tolerance for contaminants of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) there is often a need to purify and concentrate the sample solution, especially after in-gel digestion of proteins separated by two-dimensional gel electrophoresis (2-DE). A silicon microextraction chip (SMEC) for sample clean-up and trace enrichment of peptides was manufactured and investigated. The microchip structure was used to trap reversed-phase chromatography media (POROS R2 beads) that facilitates sample purification/enrichment of contaminated and dilute samples prior to the MALDI-TOF MS analysis. The validity of the SMEC sample preparation technique was successfully investigated by performing analysis on a 10 nM peptide mixture containing 2 m urea in 0.1 m phosphate-buffered saline with MALDI-TOF MS. It is demonstrated that the microchip sample clean-up and enrichment of peptides can facilitate identification of proteins from 2-DE separations. The microchip structure was also used to trap beads immobilized with trypsin, thereby effectively becoming a microreactor for enzymatic digestion of proteins. This microreactor was used to generate a peptide map from a 100 nM bovine serum albumin sample.     

Validation of a real-time RT-PCR method to quantify Newcastle Disease Virus (NDV) titer and comparison with other quantifiable methods

A method for the rapid detection and quantification of Newcastle disease virus (NDV) produced in an animal cell culture-based production system was developed to enhance the speed of the NDV vaccine manufacturing process. A SYBR Green I-based real-time RT-PCR was designed with a conventional, inexpensive RT-PCR kit targeting the F gene of the NDV LaSota strain. The method developed in this study was validated for specificity, accuracy, precision, linearity, limit of detection (LOD), limit of quantification (LOQ), and robustness. The validation results satisfied the predetermined acceptance criteria. The validated method was used to quantify virus samples produced in an animal cell culture-based production system. The method was able to quantify the NDV samples from mid- or late-production phases, but not effective on samples from the early-production phase. For comparison with other quantifiable methods, immunoblotting, plaque assay, and tissue culture infectious dose 50 (TCID50) assay were also performed with the NDV samples. The results demonstrated that the real-time RT-PCR method is suitable for the rapid quantification of virus particles produced in an animal cell-culture-based production system irrespective of viral infectivity.     

Rapid and simultaneous determination of amoxicillin, penicillin G, and their major metabolites in bovine milk by ultra-high-performance liquid chromatography-tandem mass spectrometry

A rapid, sensitive, and specific method for the determination of amoxicillin (AMO), amoxicilloic acid (AMA), amoxicillin diketopiperazine-2′,5′-dione (DIKETO), penicillin G (PEN G), benzylpenicilloic acid (BPA-1), benzylpenilloic acid (BPA-2), and benzylpenillic acid (BPA-3) in bovine milk using ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) was developed and validated. The method used penicillin V (PEN V) as the internal standard and ethanol for the deproteinisation of bovine milk. Chromatographic separation of the components was performed on a Waters Acquity UPLC^® HSS T3 column (100 mm × 2.1 mm, 1.8 μm) using a mixture of 0.15% formic acid in water with 5 mM ammonium acetate and acetonitrile as the mobile phase. Gradient elution was performed at a flow rate of 0.25 mL min^?1. The mass spectrometer was operated in the positive electrospray ionisation MS/MS mode. The method was fully validated according to EU requirements, including linearity, precision, trueness, limit of quantification, limit of detection, and specificity. The results were within the ranges specified. The established method was successfully applied in the determination of AMO, PEN G, and their major metabolites in 40 commercial bovine milk samples. The results showed that 8 samples were contaminated with BPA-1 or BPA-2. The mean levels (occurrence) of BPA-1 and BPA-2 in positive samples were 287 (50%) and 320 (100%) ng mL^?1, respectively. No sample was found to be contaminated with AMO, AMA, DIKETO, PEN G, and BPA-3. These findings could play an important role in food safety, because BPA-1 and BPA-2 metabolites pose possible health risks, although they are not included in the maximum residue limit legislation.     

Selective and sensitive liquid chromatography-tandem mass spectrometry method for the determination of levonorgestrel in human plasma

A selective, sensitive and rapid liquid chromatography-tandem mass spectrometry method for the determination of levonorgestrel in plasma was developed. An Applied Biosystems API 3000 triple quadrupole mass spectrometer set to multiple reaction monitoring (MRM) mode, using atmospheric pressure photospray ionisation (APPI) in the positive mode. Using 17-alpha-methyltestosterone as internal standard (IS), liquid-liquid extraction was followed by reversed phase liquid chromatography using a phenyl-hexyl column and tandem mass spectrometric detection. The mean recovery for levonorgestrel and 17-alpha-methyltestosterone was 99.5 and 62.9%, respectively. The method was validated from 0.265 to 130 ng levonorgestrel/ml plasma with the lower limit of quantification (LLOQ) set at 0.265 ng/ml. This assay method makes use of the increased sensitivity and selectivity of tandem mass spectrometric (MS/MS) detection, allowing for a rapid (extraction and chromatography) and selective method for the determination of levonorgestrel in human plasma. The assay method was used in a pharmacokinetic study to quantify levonorgestrel in human plasma samples generated after administrating a single oral dose of 1.5 mg levonorgestrel to healthy female volunteers for up to five half lives. The total chromatographic runtime of this method was 5.0 min per sample, allowing for analysis of a large number of samples per batch.     

Ultra sensitive determination of limaprost, a prostaglandin E1 analogue, in human plasma using on-line two-dimensional reversed-phase liquid chromatography-tandem mass spectrometry

A highly sensitive and selective method has been developed and validated to determine limaprost, a prostaglandin (PG) E(1) analogue, in human plasma by on-line two-dimensional reversed-phase liquid chromatography-tandem mass spectrometry (2D-LC/MS/MS) due to the lack of efficient methods to determine very low levels of limaprost in plasma. Limaprost and its deuterium derivatives, used as internal standard, were extracted by protein precipitation and following three-step solid phase extractions. After extraction procedure, samples were analyzed by on-line 2D-LC/MS/MS with electrospray ionization in negative mode. The 2D-LC system consists of Phenyl column at first dimension and ODS at second dimension with a trapping column placed between the separation columns. The linear dynamic range of this method was 0.1-10 pg/ml with 3 ml of plasma (r >0.9987). Acceptable precision and accuracy were obtained over the calibration curve ranges. The assay has been successfully used in analyses of human plasma samples to support clinical pharmacokinetics studies.     

Mass Spectrometry Contamination from Tinuvin 770, a Common Additive in Laboratory Plastics

The superior sensitivity of current mass spectrometers makes them prone to contamination issues, which can have deleterious effects on sample analysis. Here, bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate (marketed under the name Tinuvin 770) is identified as a major contaminant in applications using liquid chromatography coupled with mass spectrometry (LC-MS). Tinuvin 770 is often added to laboratory and medical plastics as a UV stabilizer. One particular lot of microcentrifuge tubes was found to have an excess of this compound that would leach into samples and drastically interfere with LC-MS data acquisition. Further analysis found that Tinuvin 770 readily leached into polar and nonpolar solvents from the contaminated tube lot. Efforts to remove Tinuvin 770 from contaminated samples were unsuccessful. A prescreening method using MALDI-TOF MS is presented to prevent system contamination and sample loss.     

Characterization and validation of a chemiluminescent assay based on a 1,2-dioxetane for rapid detection of enterococci in contaminated water and comparison with standard methods and qPCR

Aims: To evaluate the potential for using a novel chemiluminescence‐based enzyme assay for rapid detection of enterococci in water contaminated with faecal waste. Methods and Results: The novel assay (EntLight) was based on the enzymatic hydrolysis of the chemiluminescent 1,2‐dioxetane [(4‐methoxy‐4(3‐β‐d ‐glucoside‐4‐chlorophenyl)]spiro[1,2‐dioxetane‐3‐1,3‐tricyclo[7·3·1·0^2,7]tridec‐2,7‐ene] specific for β‐d ‐glucosidase. The specificity of the proposed EntLight assay was characterized using 26 different Enterococcus strains and 10 bacterial genera other than Enterococcus. With an analysis time of ≤8 h, the assay was found to be sensitive and specific. Validation experiments were carried out using water samples contaminated with raw municipal wastewater in comparison with qPCR and ISO standard methods. EntLight was successfully applied to detect enterococci in contaminated water within ≤8 h, and the proposed assay correlated well with both qPCR and ISO standard methods (R² > 0·776). Conclusions: EntLight can be applied to rapid and simple detection of viable enterococci in water contaminated with faecal matter. Significance and Impact of the Study: The novel EntLight assay and qPCR have the potential to be used as methods for early warning (1–7 h) of faecal pollutions in different water types.     

Simultaneous quantitative analysis of methyl salicylate,ethyl salicylate and salicylic acid from biological fluids using gas chromatography–mass spectrometry

A gas chromatographic–mass spectrometric (GC–MS) assay was developed for the quantitative analysis of methyl salicylate (MeS), ethyl salicylate (ES) and salicylic acid (SA) from biological fluids. The method was validated from 100-μl rat liver homogenate preparations (5 mg/ml protein) in 70 mM KH₂PO₄ (pH 7.4) buffer and from 100 μl rat plasma. The samples were extracted with chloroform, derivatized with BSTFA and quantitated by GC–MS in the SIM mode. The standard curves ranged from 31 ng/ml to 800 or 1250 ng/ml. Relative standard deviations and bias were less than 11% in plasma and homogenate for all compounds except SA which evidenced greater variability. The assay was used in preliminary experiments to characterize the pharmacokinetics of MeS in rats.     

A simple methodological approach for counting and identifying culturable viruses adsorbed to cellulose nitrate membrane filters

We identified conditions under which Buffalo green monkey cells grew on the surfaces of cellulose nitrate membrane filters in such a way that they covered the entire surface of each filter and penetrated through the pores. When such conditions were used, poliovirus that had previously been adsorbed on the membranes infected the cells and replicated. A plaque assay method and a quantal method (most probable number of cytopathic units) were used to detect and count the viruses adsorbed on the membrane filters. Polioviruses in aqueous suspensions were then concentrated by adsorption to cellulose membrane filters and were subsequently counted without elution, a step which is necessary when the commonly used methods are employed. The pore size of the membrane filter, the sample contents, and the sample volume were optimized for tap water, seawater, and a 0.25 M glycine buffer solution. The numbers of viruses recovered under the optimized conditions were more than 50% greater than the numbers counted by the standard plaque assay. When ceftazidime was added to the assay medium in addition to the antibiotics which are typically used, the method could be used to study natural samples with low and intermediate levels of microbial pollution without decontamination of the samples. This methodological approach also allowed plaque hybridization either directly on cellulose nitrate membranes or on Hybond N+ membranes after the preparations were transferred.