High-performance liquid chromatographic assay for N2-[5-(hypoxanthin-9-yl)pentyloxycarbonyl]-L-arginine (ST 789) in plasma by cyclization with benzoin and fluorimetric detection.
This paper describes a new highly sensitive assay for N2-[5-(hypoxanthin-9-yl)pentyloxycarbonyl]-L-arginine, an immunomodulatory agent, required for clinical pharmacokinetic investigation. A pre-column derivatization by cyclization with benzoin in aqueous medium produces the fluorescent 2-substituted amino-4,5-diphenylimidazole fluorescing at 450 nm (excitation wavelength 310 nm). L-Arginine-acetyl-L-carnitinamide chloride (ST 857, II), another arginine derivative, was used as an internal standard. A C18 DB column (5 microns, 250 mm x 4.6 mm I.D.) and a 45:55 (v/v) mixture of 0.05 M ammonium phosphate at pH 7.2 and methanol as mobile phase were used. Linearity was ascertained in the range 5-100 ng. Extraction recovery from plasma proved to be higher than 90% in the range 5-50 ng/ml. Intra-day precision, expressed as coefficient of variation, was in the range 4.7-6.0%. The limit of quantification proved to be 5 ng/ml and the limit of detection 2 ng/ml at a signal-to-noise ratio of 5. The method is specific.
High-performance liquid chromatography of guanidino compounds using benzoin as a pre-column fluorescent derivatization reagent
Abstract A high-performance liquid chromatographic method is described for the simultaneous separation of nine guanidino compounds of biological importance. Guanidino compounds are converted into the corresponding fluorescent derivatives by reaction with benzoin, and separated within 25 min on a reversed-phase column, μBondapak Phenyl, with linear gradient elution using aqueous methanol containing a Tris—hydrochloric acid buffer (pH 8.5). The method is simple, rapid and sensitive; the lower limits of detection for the guanidino compounds are 20–100 fmol in a 100-μl injection volume.
Genotypic diversity in field isolates of Babesia bovis from cattle with babesiosis after vaccination.
OBJECTIVE
To determine whether particular genotypes of Babesia bovis were common to field isolates obtained from cattle properties in Queensland where the B bovis vaccine had apparently failed.


DESIGN
A comparative study of polymerase chain reaction genotypes in different populations of B bovis.


PROCEDURE
Two polymerase chain reaction assays were applied to analyse DNA extracts of B bovis vaccine (K, T and Dixie strains) and 27 field isolates from 24 properties where disease outbreaks had occurred despite the use of the vaccine. To evaluate the stability of the genotypes identified, 11 of the field isolates were inoculated into experimental cattle that had either been previously vaccinated with T strain or not vaccinated.


RESULTS
No particular genotype of B bovis was responsible for the problems observed in previously vaccinated herds. None of the isolates had genotypes identical to the vaccine strains used. No geographic trends among the genotypes were observed. Isolates that originated from the same property also had different genotypes. Blood passage of the 11 field isolates in either previously vaccinated or nonvaccinated cattle did not alter the original genotype.


CONCLUSION
No particular genotypes identified by the Bv80 and BvVA1 polymerase chain reaction assays could be associated with vaccine failures.