LC-MS-MS experiences with internal standards

Abstract

SummaryOver the years of chromatographic evolution, the purpose of using internal standards for quantitative determinations in bioanalysis has shifted from covering (correcting for) random and systematic errors of the entire method (including sample preparation, chromatography and detection) to currently covering (correcting for) random and systematic errors of the detection, by MS-MS especially. However, the need for internal standards in current LC-MS-MS is also dependent on the degree of chromatographic separation and the degree of sample clean-up. This emphasizes the need for integrated method development, including selection of an internal standard. Available options for internal standard selection are (1) no internal standard, (2) a structurally related compound, (3) a structurally similar compound or (4) a stable isotope labelled compound. An overview of the purposes of internal standards in bioanalytical LC-MS-MS is given and this overview is supported by various illustrations using different types of internal standards.An overview of the purposes of internal standards in bioanalytical LC-MS-MS is given. It is supported byvarious illustrations using different b, pes of internal standards.The option of using cleuterated internal standards has given us surprising results in some cases, where chromatographic and/or extraction behaviour of cleuterated internal standards was different from the analyte.

Intravesical Chemotherapy of High-Grade Bladder Cancer with HTI-286, A Synthetic Analogue of the Marine Sponge Product Hemiasterlin

“…Formic acid (0.05%) was present in the gradient and reequilibration segments, and column temperature was 30jC. Luteolin (Sigma), a flavenoid with similar retention time to HTI-286, was chosen as internal standard to normalize for extraction variances or potential ion suppression (25). Retention times for HTI-286 and luteolin were both f7.7 min, and multireaction monitoring in ES+ mode was used for the quantitative analysis.…”

Evaluation of minimal 13C-labelling for stable isotope dilution in organic analysis

“…The proposed multiple linear regression procedure was evaluated by comparing the results obtained for the determination of phenol in the Certified Reference Material NIST 1584 (Priority Pollutants in methanol) by GC-EI-MS using the classical isotope dilution calibration procedure and the new procedure based on multiple linear regression [applying eqn (10)]. Both 13 C 1 -labelled and 13 C 6 -labelled phenol were employed in both calibration strategies to study the influence of the number of 13 C atoms (isotope effects) on the final analyte concentration.…”

“…When minimal labelling is selected and no isotopic effects are expected the same multiple linear regression procedure [eqn (10)] can be used here. For this purpose, the measurement of at least 2 m/z is required.…”

“…3 However, when multiple labelling of a molecule is performed, differences in its physicochemical properties in comparison with the natural abundance compound have been observed. [8][9][10][11] This is particularly true with deuterated compounds, because of slightly different hydrogen bonding capabilities, resulting in slightly different extraction and derivatisation yields from those observed for the natural abundance compounds. 8 Also, undesired changes in retention times both in GC 9 and LC 10 separations have been reported.…”

“…10,11 In these situations, a minimal labelling (e.g. a single 13 C label in the molecule) might be useful as no isotopic effects or changes in retention time are expected.…”