PubMed · 10558043
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A Paxton. 1999. Stamping out specimen collection errors.. https://pubmed.ncbi.nlm.nih.gov/10558043/
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A bioanalytical method using automated sample transferring, automated solid phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS-MS) was developed for morphine (MOR), and its metabolites morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) in human plasma. Samples of 0.25 ml were transferred into 96-well plate using automatic liquid handler (Multiprobe II). Automated SPE was carried out on a 96-channel programmable liquid handling workstation (Quadra 96) using a C(18) sorbent. The extract was injected onto a silica column using an aqueous-organic mobile phase. The chromatographic run time was 3.5 min per injection, with retention times of 1.5, 2.0 and 2.6 min for MOR, M6G, and M3G, respectively. The detection was by monitoring MOR at m/z 286-->152, M6G and M3G at m/z 462-->286. The deuterated internal standards were monitored at m/z 289-->152 for MOR-d(3), and m/z 465-->289 for M6G-d(3) and M3G-d(3). The standard curve range was 0.5-50 ng ml(-1) for MOR, 1.0-100 ng ml(-1) for M6G, and 10-1000 ng ml(-1) for M3G. The inter-day precision and accuracy of the quality control samples were <8% relative standard deviation (RSD) and <7% relative error (RE) for MOR, <5% RSD and <2% RE for M6G, and <2% RSD and <4% RE for M3G.
To evaluate the performance of an automated specimen search system in the detection of caliciviruses such as Norwalk-like viruses and Sapporo-like viruses, a suitable negative staining method was developed and the viruses were examined using the system installed in a transmission electron microscope (TEM). Clear images of the viruses were obtained by staining with 2% uranyl acetate at pH 4.0 as compared with 2% phosphotungstic acid staining at any pH. When the image parameter of 30+/-6 nm for the diameter of a single virus-like particle of 2% uranyl-acetate-stained Norwalk-like virus was set on the automated specimen search system, 95% of the virus-like particles that were counted by the conventional TEM technique were detected. The system was used to detect Norwalk-like viruses in five semipurified stool samples in which Norwalk-like viruses had already been detected by reverse transcription-polymerase chain reaction assay and conventional electron microscopy. The positive detection rate for Norwalk-like viruses, which had been counted by the conventional technique, ranged from 56.2 to 77.9% using this system. Our findings indicate that the automated specimen search system installed in a TEM is suitable for the detection of caliciviruses in semipurified stool samples. The system is useful for clinical diagnosis without the need for operator intervention.
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