Biomedical subjects

R T Ambrose

Publications and source records attributed to R T Ambrose.

5 recordsLinked to original sources

PubMed1990

Candidate reference method for determining creatinine in serum: method development and interlaboratory validation.

We describe a "high-performance" liquid chromatographic (HPLC) method for accurately determining creatinine in serum. After prechromatographic precipitation of protein, we performed isocratic ion-exchange chromatography with ultraviolet detection (234 nm). Analytical results showed linearity up to 1770 mumol/L, a detection limit of 22 mumol/L, an average analytical recovery of 101%, and a CV ranging from 3% to 11%. We used certified human serum (National Institute of Standards and Technology), and additional lyophilized serum pools also assayed by definitive isotope-dilution mass spectrometry, to validate the accuracy of the HPLC method. In addition, the isocratic HPLC results showed close agreement with those obtained with a step-gradient HPLC method. We also compared the isocratic HPLC method with alkaline picrate and enzymatic methods. Our findings with samples from nonuremic, uremic, and diabetic ketoacidotic patients confirmed the positive bias previously reported with the alkaline picrate method. Interlaboratory transferability of the method was demonstrated with various commercial instruments and analytical columns. We evaluated column stability and possible interference from endogenous or exogenous compounds. On the basis of our analytical findings, we recommend the isocratic HPLC method as a candidate Reference Method for determining creatinine in serum.

Chemistry, Clinical↗

PubMed1983

Creatinine determined by "high-performance" liquid chromatography.

We describe a "high-performance" liquid-chromatographic procedure for determination of creatinine in serum. Creatinine is separated from other species by cation-exchange chromatography. Absorbance of the creatinine in the eluate is monitored at 234 nm. Results from this procedure on sera correspond closely to those of a manual Jaffé-reaction procedure involving use of a protein-free filtrate with additional cleanup of the sample with Lloyd's reagent. None of the 21 endogenous and exogenous substances that we screened interfered. The overall CV at 138 mumol of creatinine per liter is 1.35% and at 539 mumol/L is 0.72%.

Chromatography, High Pressure Liquid↗

PubMed1983

Quantitative liquid-chromatographic estimation of bilirubin species in pathological serum.

Earlier we described a "high-performance" liquid-chromatographic procedure for separating the four distinct fractions of bilirubin (unconjugated, monoconjugated, diconjugated, and protein-bound) in pathological human serum (J. Chromatogr. 226: 391-402, 1981). We have modified the prechromatography precipitation of the serum globulins required in that method and have measured the bilirubin content of the precipitate spectrophotometrically. On average, the precipitate contained less than 10% of the total bilirubin in the serum samples. Adding the value obtained for the precipitate to that obtained by chromatography for the individual bilirubin fractions gave an estimate of the concentration of the total bilirubin in the sample. For 357 samples from 132 patients, this total value correlated well with that obtained by the Jendrassik-Gróf diazo procedure (slope = 1.00; r = 0.995, linear least-squares fit). The CV for the total and fractional bilirubin measurements was, on average, less than or equal to 5% for pathological sera. Serum sampled at different times from the same patient showed significant changes in the distribution of bilirubin among the four fractions.

Bilirubin↗

PubMed1982

Isolation and preliminary characterization of a fraction of bilirubin in serum that is firmly bound to protein.

We have isolated from pathological sera a bilirubin fraction (delta) that is very tightly, if not covalently, bound to protein, most likely albumin. This delta fraction absorbed at a lambda max of 433 nm in the visible spectrum, between the lambda max of unconjugated (alpha) and that of conjugated (Bc) bilirubin when measured in solutions containing albumin. However, unlike the other bilirubin species, this fraction could not be separated from the proteins in serum by exhaustive ultrafiltration in the presence of caffeine/benzoate solution. In the Jendrassik-Grof diazo procedure for bilirubin analysis, the delta fraction gave a large direct reaction (76-89% of the total reaction). Yet, when relatively hydrophobic azo dyes were formed by reaction of the delta fraction with the diazonium salt of dichloroaniline, only 50% of the dyes were extractable from aqueous solution. On chromatography the rest remained associated with protein. Of the extractable dye, more than 70% was accounted for by two liquid-chromatographic peaks with retentions identical with those of azo dyes formed from unconjugated bilirubin. This delta fraction was not appreciably separated from protein by treatment with strong acid or base, or by prolonged digestion with various enzymes. Finally, in a highly denaturing solvent (urea/mercaptoethanol), this fraction was not dialyzable through a membrane with a 12 000-dalton cutoff.

Bilirubin↗

PubMed1981

Separation of bilirubin species in serum and bile by high-performance reversed-phase liquid chromatography.

A high-performance, reversed-phase liquid chromatographic (HPLC) procedure has been developed for the separation of at least three major bilirubin fractions in bile and four fractions in human serum. This procedure was unlike most others, in that serum was not totally deproteinized prior to injection onto the HPLC column; instead, serum was treated with an excess of sodium sulfate solution to precipitate primarily proteins larger than albumin. Injection of the filtered and diluted supernatant onto a reversed-phase column then resulted in the separation of the bilirubin species in a 24-min gradient elution run. Both the initial aqueous acidic mobile phase and the final isopropyl alcohol-based mobile phase contained 5% methoxyethanol (v/v) to facilitate elution of albumin still present in the treated sample. Bilirubin species eluting from the column were detected by absorbance at 450 nm. Results of a number of chromatographic separations of pathological sera indicated a wide variation in the relative proportions of the four bilirubin fractions observed. A correlation of the sum of the areas of the bilirubin peaks observed by HPLC was found with the total bilirubin value obtained by a standard reference procedure.

Animals↗