Measurement of Estradiol in Human Serum by LC-MS/MS Using a Novel Estrogen-Specific Derivatization Reagent

Abstract

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described that employs a novel derivatization reagent for the measurement of serum estradiol (E2), with simultaneous analysis of underivatized testosterone (T) and dihydrotestosterone (DHT). The main advantage of the new derivatization reagent 1,2-dimethylimidazole-5-sulfonyl chloride is its analyte-specific fragmentation that enables monitoring of confirmatory mass transitions with high sensitivity. The reaction mixture can be analyzed without additional purification steps using a 9.5 min gradient run, and sensitive detection is achieved with a triple quadrupole mass spectrometer using atmospheric pressure photoionization. Method validation was performed with human serum samples, including a comparison with a standard LC-MS/MS method using 120 samples from a clinical study, and analysis of certified E2 serum reference materials BCR-576, BCR-577, and BCR-578. The lower limits of quantification for E2, T, and DHT were 0.5 pg/mL, 25 pg/mL, and 0.10 ng/mL, respectively, from a 200-μL sample. Validation results indicated good accuracy and agreement with established, conventional LC-MS/MS assays, demonstrating suitability for analysis of samples containing E2 in the low pg/mL range, such as serum from men, children, and postmenopausal women.

High-Sensitivity Micro LC-MS/MS Assay for Serum Estradiol without Derivatization

“…Derivatization of E 2 has been widely accepted as a way to improve ionization efficiency, making it more sensitive in LC-MS/MS-based detection. Most reported methods of derivatization use dansyl chloride and found improved signal intensity by 1-2 orders of magnitude when compared to an underivatized method under positive polarity mode (23)(24)(25)(26)(27)(28). However, the derivatization step can add complexity in the sample preparation and may decrease the robustness of the overall method, which is not optimal for a routine clinical laboratory application.…”

“…To overcome these technical limitations of immunoassays, mass spectrometry assays, especially LC-MS/MS assays, have increasingly been used in the recent years. One drawback about most LC-MS/MS E 2 assays is that derivatization is required in the sample preparation procedure to achieve sufficient sensitivity (4,9,(25)(26)(27). In a routine clinical laboratory, derivatization can increase the sample preparation time and introduce more method variations.…”

Quantitative analysis of endogenous compounds

“…Various approaches ( Figure 2) are followed to address the lack of blank matrices for the quantification of endogenous compounds by LC-MS/MS including, background subtraction [26-31], the standard addition method [24,25,[32][33][34][35][36][37][38][39][40][41][42][43][44], neat solutions [45][46][47][48][49][50][51][52], artificial matrices of biological fluids [23,, stripped matrices [8,10,11,[74][75][76][77][78][79][80][81][82][83][84][85][86][87][88][89][90][91], and surrogate analytes [66,[92][93][94][95][96][97]…”

Simultaneous measurement of total estradiol and testosterone in human serum by isotope dilution liquid chromatography tandem mass spectrometry

“…In addition, a mobile phase additive of ammonium fluoride was used to further improve E2 negative ionization efficiency as described previously [24, 46]. Several LC-MS/MS methods for E2 or steroid profiles use dansyl chloride as a derivatizing agent to enhance the ionization and thus to achieve higher sensitivity (46–49). However, the dansyl fragment ion is used to quantitate E2 with these methods, therefore limiting the specificity of the method.…”