Immunoaffinity isolation of the sulfate conjugate of 4'-hydroxypropranolol from plasma.

Selective extraction of sulfate conjugates of basic drugs from biological matrices has been difficult because of their highly polar nature. Immunoaffinity isolation may be the best solution to this analytical problem. This was tested for a model compound, the metabolite 4'-hydroxypropranolol sulfate (HOPS), which was effectively extracted from plasma by a column containing antibodies to the parent drug propranolol. The specificity was very high, giving little interference from the biological material in subsequent high-performance liquid chromatographic analysis with fluorometric detection. The method for HOPS was highly reproducible and provided a sensitivity of 1 ng/ml plasma. The technique was applied to measurements of HOPS in plasma after therapeutic doses of propranolol as well as to the individual enantiomers after chiral derivatization.

Quantitative analysis of 6-keto-prostaglandin F1 alpha using immunoaffinity purification and gas chromatography-mass spectrometry.

This paper describes an immunoaffinity purification technique for 6-keto-prostaglandin F1 alpha (6KPGF1 alpha) prior to quantitative analysis by high-resolution gas chromatography-negative-ion chemical ionization mass spectrometry (HRGC-NICIMS). Polyclonal antibodies to 6KPGF1 alpha were partially purified using Staphylococcus aureus Protein A immobilized on Sepharose CL-4B. This partially purified fraction was covalently bound to silica gel using N-hydroxysuccinimidyl-functionalized silica. Columns constructed using this gel quantitatively bound 6KPGF1 alpha which could be eluted quantitatively with acetonitrile-water (19:1). Binding capacity was reconstituted by washing with 0.01 M phosphate buffer (pH 7.4). Human urinary and canine plasma 6KPGF1 alpha was sufficiently purified using these columns that HRGC-NICIMS analysis of the methoxime-pentafluorobenzyl-tris-trimethylsilyl derivative was interference-free.

Extraction of thromboxane B2 from urine using an immobilized antibody column for subsequent analysis by gas chromatography-mass spectrometry.

This paper describes an antibody affinity (immunoaffinity) column which, in one step, extracts and sufficiently purifies urinary thromboxane B2 (TXB2) for quantitative analysis by high resolution gas chromatography-negative ion chemical ionization-selected ion monitoring-mass spectrometry (HRGC-NICI-SIM-MS). Polyclonal TXB2 antibody from rabbit was partially purified using immobilized Staphylococcus aureus Protein A. The purified IgG fraction was then immobilized using an N-hydroxysuccimidyl silica gel. The resulting matrix bound 570 ng TXB2 per ml of gel. TXB2 was quantitatively eluted with acetonitrile-water (19:1). Columns constructed from the gel could be used repeatedly since binding capacity was reconstituted using 0.01 M phosphate buffer (pH 7.4) with no apparent loss of activity. Using these columns, urinary TXB2 was sufficiently purified in one step such that in subsequent analysis by HRGC-NICI-SIM-MS interference free chromatograms were observed.

Quantitative analysis of adenosine: statistical comparison of radioimmunoassay and gas chromatography-mass spectrometry-selected ion monitoring methods.

A new method for quantitating adenosine concentration by capillary gas chromatography-mass spectrometry-selected ion monitoring (GC-MS-SIM) has been developed and used as a reference method for evaluating a newly developed radioimmunoassay (RIA) for adenosine. Details of the GC-MS-SIM method are presented, along with the comparative results and uncertainties of both methods. General considerations in the statistical analysis of method comparison data are discussed with particular reference to studies using quantitative mass spectrometry as the standard method; the adenosine methods are used as specific examples in this discussion. Simultaneous estimation of the y-intercept and slope of the least squares regression line relating the results of the two methods using the 95% joint confidence ellipse demonstrated the absence of either constant or proportional error between the two methods. The relatively small uncertainty in the GC-MS-SIM measurements had no significant effect on the linear regression. Random error between the two methods was detected, and was estimated by the coefficient of variation in the RIA data as ten percent of the RIA value.