PubMed · 14112672
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A HAESSIG, P HOLLANDER. 1963. [PASTEURIZED PLASMA PROTEIN SOLUTIONS].. https://pubmed.ncbi.nlm.nih.gov/14112672/
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BACKGROUND: Because mycophenolic acid (MPA) is highly protein bound and because the free fraction is the pharmacologically active portion, a rapid, reliable, and sensitive procedure is required to study the relationship between free MPA and treatment efficacy/toxicity. Liquid chromatography-tandem mass spectrometry is ideally suited for such a method. METHODS: Free MPA was isolated from plasma by ultrafiltration. An online extraction cartridge with a column-switching technique, analytical liquid chromatography over an Aqua Perfect C(18) column, and electrospray tandem mass spectrometry was used to quantify free and total MPA. To investigate ion suppression, a continuous infusion of MPA was introduced into the effluent from the HPLC column, and different ultrafiltrates and extracted plasma samples were injected on the column. RESULTS: A chromatographic run time of 4 min separated MPA from metabolites and internal standard, thereby avoiding interference from in-source fragmentation. Ion suppression occurred well before elution of MPA and internal standard. The lower limit of quantification for free MPA was 0.5 microg/L, and the method was linear to 1000 microg/L. Interassay imprecision (CV) was <10% for free MPA (0.5-333 microg/L). Agreement was good for free MPA (n = 52) and total MPA (n = 106) between the proposed method and a validated HPLC method with ultraviolet detection. The Passing-Bablok regression line was: y = 0.95x + 0.27 microg/L for free MPA and y = 0.98x + 0.03 mg/L for total MPA. CONCLUSIONS: The presented method allows the accurate, precise, and rapid determination of free and total MPA in plasma over a wide analytical range covering the concentrations relevant to pharmacokinetic studies and routine monitoring of this drug.
Postprandial plasma triglyceride (ppTG) and NEFA clearance were stratified by plasma acylation-stimulating protein (ASP) and gender to determine the contribution of fasting ASP in a normal population (70 men; 71 women). In the highest ASP tertile only, ASP decreased over 8 h (90 +/- 9.7 nM to 70 +/- 5.9 nM, P<0.05 males; 61.9 +/- 4.0 nM to 45.6 +/- 6.2 nM, P<0.01 females). Fasting ASP correlated positively with ppTG response. ppTG (P<0.0001, 2-way ANOVA, both genders) and NEFA levels progressively increased from lowest to highest ASP tertile, with the greatest differences in males. By stepwise multiple regression, the best prediction of ppTG was: (fasting ASP + apolipoprotein B + insulin + TG; r=0.806) for men and (fasting ASP + total cholesterol; r=0.574) for women. Leptin, body mass index, and other fasting variables did not improve the prediction. Thus, in men and women, ASP significantly predicted ppTG and NEFA clearance and, based on lower ASP, women may be more ASP sensitive than men. Plasma ASP may be useful as a fasting variable that will provide additional information regarding ppTG and NEFA clearance.
A semi-automatic, high-throughput method has been developed to rapidly assess plasma protein binding of new chemical entities in drug discovery phase. New chemical entities are mixed with plasma and the unbound fractions are separated from the bound fraction by ultrafiltration in a 96-well filtrate assembly. The unbound fractions are then analyzed by fast liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sample handling is automated by a robotic system. Employing a cocktail approach where multiple new chemical entities are allowed to bind to plasma proteins in the same well has further increased the throughput. We have validated the method with 12 commercially available compounds. The plasma protein binding data obtained by this method are comparable with the literature values. This method enables the determination of protein binding for 32 compounds in one single experiment instead of 1-2 compounds using the conventional methods.