Comparison of Heavy Labeled (SIL) Peptide versus SILAC Protein Internal Standards for LC-MS/MS Quantification of Hepatic Drug Transporters

We studied the precision of quantification of organic anion-transporting polypeptide 1B1 (OATP1B1), OATP1B3, OATP2B1, and P-glycoprotein (P-gp) in human livers by surrogate peptide based LC-MS/MS approach using two different internal standards: stable isotope labeled peptide (SIL) versus stable isotope labeled protein (SILAC). The SIL peptides were procured commercially and the SILAC proteins were generated in-house by labeling arginine and/or lysine residues in cells expressing these transporters. Liver tissue n=20 was homogenized and the membrane fraction was isolated. The membranes were trypsin digested and the peptides were analyzed using LC-MS/MS under optimized conditions. The precision in the quantification of proteins in three independently trypsin digested samples from each liver was calculated as the standard deviation of the log transformed protein concentration. The precision of the SIL internal standard method was either slightly (P<0.05, paired t-test) better than that of the SILAC method (OATP1B1, OATP1B3, and P-gp) or not different (OATP2B1). Trypsin digestion, as measured by the response of the labeled peptide derived from the SILAC protein, was consistent across liver samples. These results indicate that when maximum trypsin digestion is ensured, the SIL internal standard method can be used with confidence for quantification of drug transporters.

Download Full-text

Determination and Thin Layer Chromatographic Confirmation of Identity of Aflatoxins B1 and M1 in Artificially Contaminated Beef Livers: Collaborative Study

Journal of AOAC INTERNATIONAL ◽

10.1093/jaoac/65.6.1435 ◽

1982 ◽

Vol 65 (6) ◽

pp. 1435-1444 ◽

Cited By ~ 1

Author(s):

Robert D Stubblefield ◽

William F Kwolek ◽

Leonard Stoloff ◽

◽

J M Fremy ◽

...

Keyword(s):

Thin Layer ◽

Standard Method ◽

Direct Method ◽

Collaborative Study ◽

Internal Standard ◽

Test Methods ◽

Internal Standard Method ◽

Components Of Variance ◽

Internal Standards ◽

Beef Liver

Abstract An international collaborative study involving 13 laboratories was conducted to test methods for the determination and thin layer chromatographic (TLC) confirmation of identity of aflatoxins B1 and M1 in beef liver. For the determination, each collaborator furnished fresh or frozen beef liver. Samples were artificially contaminated by adding solutions containing various concentrations of aflatoxins B1 and M1 (0.032-0.69 ng/g). Two TLC confirmation methods were tested with extracts obtained from the determination. Two measurement methods using 2-dimensional TLC were evaluated. In the first, sample extracts were compared directly with B1 and M1 standards on TLC plates; in the second, internal standards plus sample extracts were compared with Bi and M1 standards on the plates. Average within-laboratory coefficients of variation (CV) for the direct method were 26% for B1 and 26% for M1 compared with 24 and 26%, respectively, for the internal standard method. The average between-laboratory CV values were 39% for Bi and 41% for M1 by the direct method and 36% for B1 and 39% for M1 by the internal standard method. Recoveries ranged from 64 to 90% for Bi and from 72 to 86% for M1. These data indicate that the more convenient direct method was sufficient, and internal standards were unnecessary. An analysis of variance was calculated from combined sample data to determine components of variance. The within-laboratory CV values were 27.0 and 32.3% for B1 and M1, respectively, and the between-laboratory CV values were 47.1 and 53.2%, respectively. Both TLC confirmation methods gave satisfactory results and have been adopted official first action, along with the determination method.

Download Full-text

Determination of alcohols in mixtures by 19F NMR spectroscopy using hexefluoroacetone reagent

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19821973 ◽

1982 ◽

Vol 47 (7) ◽

pp. 1973-1978 ◽

Cited By ~ 2

Author(s):

Jiří Karhan ◽

Zbyněk Ksandr ◽

Jiřina Vlková ◽

Věra Špatná

Keyword(s):

Standard Deviation ◽

Nmr Spectroscopy ◽

Standard Method ◽

Internal Standard ◽

Internal Standard Method ◽

Concentration Region ◽

Experimental Conditions ◽

Curve Method ◽

Sample Preparation Procedure

The determination of alcohols by 19F NMR spectroscopy making use of their reaction with hexafluoroacetone giving rise to hemiacetals was studied on butanols. The calibration curve method and the internal standard method were used and the results were mutually compared. The effects of some experimental conditions, viz. the sample preparation procedure, concentration, spectrometer setting, and electronic integration, were investigated; the conditions, particularly the concentrations, proved to have a statistically significant effect on the results of determination. For the internal standard method, the standard deviation was 0.061 in the concentration region 0.032-0.74 mol l-1. The method was applied to a determination of alcohols in the distillation residue from an oxo synthesis.

Download Full-text