Biomedical subjects

D Buhrman

Publications and source records attributed to D Buhrman.

3 recordsLinked to original sources

PubMed1999

Quantitative determination of an extremely polar compound allantoin in human urine by LC-MS/MS based on the separation on a polymeric amino column.

Quantitative determination of allantoin in biological matrix poses a challenge to bioanalysis due to the extreme polarity of allantoin. The molecule exhibits virtually no retention on any of the available hydrophobic HPLC packing materials. In this study, an assay was developed for the LC-MS/MS quantitation of allantoin in human urine using a polymeric amino column to achieve the necessary separation. The urine samples were prepared by a single-step solid phase extraction (SPE) procedure. The extracted samples were analyzed on a jordi-gel DVB polyamine column (operated under an acetonitrile/water gradient with water as the stronger mobile phase) interfaced with a Finnigan TSQ 7,000 mass spectrometer. Negative electrospray ionization (ESI) was employed as the ionization source. Allantoin and its internal standard (13C1, 15N1-allantoin) were detected by use of single reaction monitoring (SRM) mode. The method was validated in the concentration range of 14.6-213 microg/ml(-1), with within and between run accuracy and precision both < 7%. The method has been successfully applied to clinical sample analysis.

Acetonitriles↗

PubMed1998

High throughput liquid chromatography/mass spectrometry bioanalysis using 96-well disk solid phase extraction plate for the sample preparation.

The 96 well solid phase extraction (SPE) operated by robot in the LC/MS/MS bioanalysis offered rapid sample preparation for drugs and metabolites in biological matrices, based on simultaneous extraction of 96 samples. The use of a disk as sorbent in the 96 well plate further improved the performance of SPE and allowed for small elution volumes, making it possible to 'dilute and shoot" after SPE elution. In this study, a 96 well plate (Empore) was developed, characterized and optimized for several pharmaceutical compounds. In addition, a robot (MultiProbe) was modified to automate the 96 well plate operation. Examples were given to illustrate the major differences of using 96 well disk plate SPE in the method development as compared to the traditional SPE. This technology has been successfully used to support many clinical studies. Typically, a batch of 96 samples were prepared in 1-1.5 hours unattended (except for the replacement of a collection plate). Considerable savings in disposable supplies were also noted.

Anti-Asthmatic Agents↗

PubMed1991

Biological properties of RB51; a stable rough strain of Brucella abortus.

A rifampin-resistant mutant of Brucella abortus, designated RB51, was derived by repeated passage of strain 2308 on Trypticase soy supplemented with 1.5% agar and varying concentrations rifampin or penicillin. The RB51 colonies absorbed crystal violet and RB51 cell suspensions autoagglutinated, indicating a rough type colonial morphology for this strain. No O-chain component was detected in lipopolysaccharide (LPS) extracted from RB51 on SDS-PAGE gels stained with silver. Western blot analysis with the monoclonal antibody BRU 38, which is specific for the perosamine homopolymer O-chain of smooth Brucella LPS, indicated that the LPS of RB51 is highly deficient in O-chain when compared with the parenteral smooth strain 2308 or rough strain 45/20. Biochemically, RB51 resembles parental strain 2308 in its ability to utilize erythritol. Intraperitoneal inoculation of RB51 into mice results in a splenic colonization which is cleared within four weeks post infection. RB51 does not revert to smooth colony morphology upon passage in vivo (mice) or in vitro. Mice infected with RB51 produce antibodies against B. abortus antigens including class 2 and 3 outer membrane proteins but not against the O-chain. Furthermore, rabbits, goats and cattle hyperimmunized with sonicates of RB51 develop antibodies to B. abortus cellular antigens but do not develop antibodies specific for the O-chain. Immunization of mice with 1 x 10(8) viable RB51 organisms confers significant protection against challenge with virulent B. abortus strain 2308.

Animals↗