Diego F. Cobice

Derivatization of estrogens enhances specificity and sensitivity of analysis of human plasma and serum by liquid chromatography tandem mass spectrometry

Estrogens circulate at concentrations less than 20 pg/mL in men and postmenopausal women, presenting analytical challenges. Quantitation by immunoassay is unreliable at these low concentrations. Liquid chromatography tandem mass spectrometry (LC–MS/MS) offers greater specificity and sometimes greater sensitivity, but ionization of estrogens is inefficient. Introduction of charged moieties may enhance ionization, but many such derivatives of estrogens generate non-specific product ions originating from the “reagent” group. Therefore an approach generating derivatives with product ions specific to individual estrogens was sought. Estrogens were extracted from human plasma and serum using solid phase extraction and derivatized using 2-fluoro-1-methylpyridinium-p-toluenesulfonate (FMP-TS). Electrospray in positive mode with multiple reaction monitoring using a QTrap 5500 mass spectrometer was used to quantify “FMP” derivatives of estrogens, following LC separation. Transitions for the FMP derivatives of estrone (E1) and estradiol (E2) were compound specific (m/z 362→238 and m/z 364→128, respectively). The limits of detection and quantitation were 0.2 pg on-column and the method was linear from 1–400 pg/sample. Measures of intra- and inter-assay variability, precision and accuracy were acceptable (<20%). The derivatives were stable over 24 h at 10 °C (7–9% degradation). Using this approach, E1 and E2, respectively were detected in human plasma and serum: pre-menopausal female serum (0.5 mL) 135–473, 193–722 pmol/L; male plasma (1 mL) 25–111, 60–180 pmol/L and post-menopausal female plasma (2 mL), 22–78, 29–50 pmol/L. Thus FMP derivatization, in conjunction with LC–MS/MS, is suitable for quantitative analysis of estrogens in low abundance in plasma and serum, offering advantages in specificity over immunoassay and existing MS techniques.

11β-Hydroxysteroid dehydrogenase type 1 contributes to the balance between 7-keto- and 7-hydroxy-oxysterols in vivo

Graphical abstract

Measurement of tamsulosin in human serum by liquid chromatography–tandem mass spectrometry

Highlights • A method to quantify tamsulosin by liquid chromatography–tandem mass spectrometry.• Simple extraction method from serum, excellent recovery, linear range 0.2–50 ng/mL.• In-house synthesis of internal standard, d9-finasteride.• Validated method with acceptable reproducibility, precision, accuracy and stability.• Useful to assess compliance and pharmacokinetics in studies of benign prostatic hyperplasia.

Quantification of 11β-hydroxysteroid dehydrogenase 1 kinetics and pharmacodynamic effects of inhibitors in brain using mass spectrometry imaging and stable-isotope tracers in mice

Graphical abstract Figure. No caption available. ABSTRACT 11&bgr;‐Hydroxysteroid dehydrogenase 1 (11&bgr;‐HSD1; EC 1.1.1.146) generates active glucocorticoid hormones. Small molecule inhibitors have been developed to target 11&bgr;‐HSD1 for the treatment of dementia; these must enter brain subregions, such as the hippocampus, to be effective. We previously reported mass spectrometry imaging measurement of murine tissue steroids, and deuterated steroid tracer infusion quantification of 11&bgr;‐HSD1 turnover in humans. Here, these tools are combined to assess tissue pharmacokinetics and pharmacodynamics of an 11&bgr;‐HSD1 inhibitor that accesses the brain. [9,11,12,12‐2H]4‐Cortisol was infused (1.75 mg/day) by minipump for 2 days into C57Bl6 mice (male, age 12 weeks, n = 3/group) after which an 11&bgr;‐HSD1 inhibitor (UE2316) was administered (25 mg/kg oral gavage) and animals culled immediately or 1, 2 and 4 h post‐dosing. Mice with global genetic disruption of Hsd11B1 were studied similarly. Turnover of d4‐cortisol to d3‐cortisone (by loss of the 11‐deuterium) and regeneration of d3‐cortisol (by 11&bgr;‐HSD1‐mediated reduction) were assessed in plasma, liver and brain using matrix assisted laser desorption ionization coupled to Fourier transform cyclotron resonance mass spectrometry. The tracer d4‐cortisol was detected in liver and brain following a two day infusion. Turnover to d3‐cortisone and on to d3‐cortisol was slower in brain than liver. In contrast, d3‐cortisol was not detected in mice lacking 11&bgr;‐HSD1. UE2316 impaired d3‐cortisol generation measured in whole body (assessed in plasma; 53.1% suppression in rate of appearance in d3‐cortisol), liver and brain. Differential inhibition in brain regions was observed; active glucocorticoids were suppressed to a greater in extent hippocampus or cortex than in amygdala. These data confirm that the contribution of 11&bgr;‐HSD1 to the tissue glucocorticoid pool, and the consequences of enzyme inhibition on active glucocorticoid concentrations, are substantial, including in the brain. They further demonstrate the value of mass spectrometry imaging in pharmacokinetic and pharmacodynamic studies.

Future technology insight

In pharmaceutical research, understanding the biodistribution, accumulation and metabolism of drugs in tissue plays a key role during drug discovery and development. In particular, information regarding pharmacokinetics, pharmacodynamics and transport properties of compounds in tissues is crucial during early screening. Historically, the abundance and distribution of drugs have been assessed by well‐established techniques such as quantitative whole‐body autoradiography (WBA) or tissue homogenization with LC/MS analysis. However, WBA does not distinguish active drug from its metabolites and LC/MS, while highly sensitive, does not report spatial distribution. Mass spectrometry imaging (MSI) can discriminate drug and its metabolites and endogenous compounds, while simultaneously reporting their distribution. MSI data are influencing drug development and currently used in investigational studies in areas such as compound toxicity. In in vivo studies MSI results may soon be used to support new drug regulatory applications, although clinical trial MSI data will take longer to be validated for incorporation into submissions. We review the current and future applications of MSI, focussing on applications for drug discovery and development, with examples to highlight the impact of this promising technique in early drug screening. Recent sample preparation and analysis methods that enable effective MSI, including quantitative analysis of drugs from tissue sections will be summarized and key aspects of methodological protocols to increase the effectiveness of MSI analysis for previously undetectable targets addressed. These examples highlight how MSI has become a powerful tool in drug research and development and offers great potential in streamlining the drug discovery process.

Future technology insight: mass spectrometry imaging as a tool in drug research and development: MS imaging for future drug development

In pharmaceutical research, understanding the biodistribution, accumulation and metabolism of drugs in tissue plays a key role during drug discovery and development. In particular, information regarding pharmacokinetics, pharmacodynamics and transport properties of compounds in tissues is crucial during early screening. Historically, the abundance and distribution of drugs have been assessed by well‐established techniques such as quantitative whole‐body autoradiography (WBA) or tissue homogenization with LC/MS analysis. However, WBA does not distinguish active drug from its metabolites and LC/MS, while highly sensitive, does not report spatial distribution. Mass spectrometry imaging (MSI) can discriminate drug and its metabolites and endogenous compounds, while simultaneously reporting their distribution. MSI data are influencing drug development and currently used in investigational studies in areas such as compound toxicity. In in vivo studies MSI results may soon be used to support new drug regulatory applications, although clinical trial MSI data will take longer to be validated for incorporation into submissions. We review the current and future applications of MSI, focussing on applications for drug discovery and development, with examples to highlight the impact of this promising technique in early drug screening. Recent sample preparation and analysis methods that enable effective MSI, including quantitative analysis of drugs from tissue sections will be summarized and key aspects of methodological protocols to increase the effectiveness of MSI analysis for previously undetectable targets addressed. These examples highlight how MSI has become a powerful tool in drug research and development and offers great potential in streamlining the drug discovery process.