Data for analysis of catechol estrogen metabolites in human plasma by liquid chromatography tandem mass spectrometry

Analysis of catechol estrogens (2 & 4 hydroxy-estrone and estradiol) has proven troublesome by liquid chromatography tandem mass spectrometry due to their low concentrations, short half-lives and temperature-labile nature. Derivatization to methyl piperazine analogues has been reported for a panel of 9 estrogens in, “Derivatization enhances analysis of estrogens and their bioactive metabolites in human plasma by liquid chromatography tandem mass spectrometry” (Denver et al., 2019). Data show alteration of the base catalyst in this method was required to allow detection of catechol estrogens to low levels. Data also highlight the challenges faced in chromatographic separation of isomers and isotopologues, which were partially overcome by employing an extended column length and reduced oven temperature. In addition, data analysis displayed significant matrix effects during quantitation in plasma, following solid-phase extraction, despite efficient recoveries.


Data
Here we display data in Table 1, which illustrates the mass spectrometry tuning parameters of MPPZ-derivatives of catechol estrogens, shown by their exact theoretical and observed masses. In Table  2, data demonstrating the limit of detection that can be achieved for analytes following derivatization are given for unextracted standards, alongside observed retention times from chromatographic interpretation, Fig. 1. Finally, the recoveries of catechol estrogens from plasma following solid-phase extraction (SPE) are displayed in Table 2, with associated data describing matrix effects.
Catechol estrogens (2 and 4-hydroxy-estrogens) are challenging metabolites to analyze by LC-MS/ MS [2,3]. Common analytical challenges arise due to their unstable nature and short half-lives [4]. Derivatization to 1-(2, 4-dinitrophenyl)-4,4-dimethylpiperazinium (MPPZ) derivatives has been successfully applied for analysis of estrone, estradiol, 16-hydroxy and 2 and 4 methoxyestrogens [1]. Here the successes and pitfalls of applying this approach to analyse catechol estrogens are described. For 2 &4-hydroxyestrogens, MPPZ derivatives were generated with the original protocol [1] but with poor yield, with insufficient detection upon lowering the concentrations (<500 pg mL À1 ). Comparison of various derivatization base catalysts (sodium bicarbonate, triethylamine, pyridine, ammonium hydroxide and N-diethylaniline) was key in achieving efficient derivatization. Modification of the base catalyst to N-diethylaniline enhanced PPZ derivatization with catechol estrogens, showing Â500 increase in peak area response, but this approach caused reduction of signals of the 9 other estrogens (E1, E2, 16-hydroxy and 4 and 2-methoxy-estrogens) within this sex steroid pathway. Combinations of base catalysts were also tested to create a holistic approach. However, derivatization of the catechol metabolites and additional 9 estrogens were only successful in separate reactions.
Structural identification of precursor and product ions for the catechol derivates were achieved by high resolution MS and multiple reaction monitoring for quantitation established by triple quadrupole MS, Table 1.
Double derivatives of these compounds were not seen, but isomeric mono-derivatives were observed, believed due to the possibility of either of the A-ring hydroxyl groups reacting. To achieve the highest degree of chromatographic resolution of isomers and isotopologues, a C18_PFP (2.1 Â 150mm) Specifications  Value of the data Illustrates a common problem faced in quantitative estrogen metabolite assays for catechol estrogens LC-MS/MS parameters are reported for identification and resolution of catechol metabolites The derivatization method allows analyte detection to 20 pg mL À1 in aqueous solutions Recovery and ion suppression data for researchers considering solid phase extraction of these analytes was coupled to a C18_PFP (2.1 Â 20mm). The reduction of oven temperature from 25 to 20 C also aided in resolving the catechol estrogen derivative peaks (Fig. 1). Data illustrated that recovery of catechol estrogens (pre vs post-spiked PA) from Oasis MCX cartridges, Table 2    Hydroxyestrone ( 13 C 6 -4OHE1) and 13,14,15,16,17,18-13 C 6 -2-hydroxyestradiol ( 13 C 6 -2OHE2) were from CK Isotopes Limited (Leicestershire, UK). N-Diethylaniline was from Acros Organics (Geel, Belgium). All additional reagents were sourced as specified in Denver et al. [1].

Methods
Analysis, including assessment of extraction efficiency and ion suppression, was performed according to the approach described in Denver et al. [1] and modifications for catechol estrogens reported below.

Instrumentation
Structures of fragment ions formed from estrogen derivatives were determined by high resolution MS using a SYNAPT G2Si instrument (Waters Corp, Manchester, UK) fitted with an ESI source in positive mode [1]. Method development was performed using a Shimadzu Nexera X2 LC (Shimadzu, Kyoto, Japan) coupled to a Sciex 6500 þ Mass Spectrometer (Sciex, Warrington, UK) operated in positive electrospray (ESI).

Chromatographic conditions
Estrogen metabolites were analyzed both individually and in a mixed solution to confirm separation. Two Ace Excel 2 C18-PFP column (150 Â 2.1 mm, 2 mm þ 20 Â 2.1 mm, 2 mm; HiChrom, Reading, England) were coupled at an oven temperature of 20 C. A gradient solvent system of water: acetonitrile (90:10), containing formic acid (FA; 0.1%, 0.5 mL/min) was diverted to waste for the initial 9 minutes followed by elution for a further 4 minutes at 90:10, then with a gradient over 3 minutes until final conditions of water: acetonitrile (90:10) containing FA (0.1%, 0.5 mL/min) were achieved. Injection volume was 30 mL.

Derivatization and optimization
The following protocol was applied for derivatization PPZ stock (10 mL; 1mg mL À1 ), N-diethylaniline (10 mL) and acetone (70 mL) were added to the catechol estrogen standards and was capped and incubated (60 C, 1 h). Reagents were reduced to dryness at 40 C under oxygen free nitrogen (OFN).

Extraction and optimization
SPE using Oasis ® MCX (3 cc/60 mg, Waters, Wilmslow, UK) cartridges was applied under gravity. Prior to loading the sample, cartridges were conditioned and equilibrated with methanol (2 mL), followed by water (2 mL). The diluted sample (0.5 mL plasma þ 0.5mL water (or 1 mL water for standards) þ 200 pg mL À1 Internal Standard) was loaded and allowed to pass through the cartridges and the eluate discarded. The cartridges were washed with aqueous FA (2% v/v, 2 mL). A second wash of methanol (60% v/v, 2 mL) was applied with the eluate discarded. Steroids were eluted in methanol (95%; 2 mL). Extracts were reduced to dryness under OFN (40 C) and the residues were derivatized as above.