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Analysis of estradiol and analytes with phenolic OH using labeling chemistry and LC-MSMS workflow

An analyte, phenolic technique for the analysis of estradiol and analytes with phenolic OH using labeling chemistry and LC-MSMS workflows

Active Publication Date: 2014-09-10
DH TECH DEVMENT PTE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, quantification of these molecules, such as estradiol, in biological matrices can be difficult, especially at low concentrations
[0009] Although reagents such as pentafluorobenzyl bromide and dansyl chloride are commonly used in derivatization strategies for estradiol analysis using mass spectrometry, current LC / MSMS strategies involving such derivatization of analytes containing phenolic OH The strategy failed to achieve the detection limits required for clinical analysis (Shimada et al., Analyst, 116, 1393-1397 (1991); Higashi et al., Chem. Pharm. Bull.) 54(11), 1479-1485(2006))

Method used

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  • Analysis of estradiol and analytes with phenolic OH using labeling chemistry and LC-MSMS workflow
  • Analysis of estradiol and analytes with phenolic OH using labeling chemistry and LC-MSMS workflow
  • Analysis of estradiol and analytes with phenolic OH using labeling chemistry and LC-MSMS workflow

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0162] Example 1-Illustrated steps for the synthesis of labeled QAOC reagents

[0163]

[0164]

[0165] Synthesis of 4-acetyl-4-phenyl-1-carboethoxysemicarbazide (1)

[0166] A solution of 4-acetylphenylisocyanate (10 g, 62.05 mmol) in toluene (250 mL) was added dropwise to a solution of ethyl carbazate (6.46 g, 62.05 mmol) in toluene (100 mL). The reaction mixture was stirred at room temperature for 2 h and then at 80 °C for 2 h. The precipitate formed in the reaction was filtered and dried in a vacuum oven to give p-acetyl-4-phenyl-1-carboethoxysemicarbazide 1 (16.5 g, 90%). It was used in the next reaction step without further purification. (Synthesis adapted from: Organic Syntheses (Organic Syntheses, Coll.) Volume 6, page 936 (1988); Volume 51, page 121 (1971). 4-phenyl-1,2,4-triazole phenoline-3,5-dione). In various aspects, the value of each ingredient used in this example can be increased or decreased by an amount in the range of about 5% to about 20%, for e...

example 2

[0174] Example 2 - Sample Enrichment

[0175] Sample volume, extraction solvent and its composition, reagent solutions, amount of extraction medium and molecular weight cut-off membrane type are typical.

[0176] Dried blood spots: Dried human or animal blood samples (3-10 μL) on filter paper were spiked with internal standard and spiked with 100 μL ethyl acetate-hexane, preferably in a 1:1 composition, more preferably multiple times Extraction is performed efficiently (sonication may be required to improve extraction efficiency). The combined ethyl acetate-hexane layers were then dried using a centrifugal vacuum concentrator or a hot gas stream or a combination of both. Add 50 μL of reagent solution (2 mg / mL in acetonitrile) to dry samples. Samples were vortexed to mix and then incubated at room temperature for at least 30 min. Water (20 μL) was added, samples were vortexed to mix, and samples were then analyzed by LC-MSMS.

[0177] Protein precipitation: Add 0.8 mL of ac...

example 3

[0181] Example 3 - Mass spectrometry

[0182] Samples can be analyzed using the following parameters in LC-MSMS analysis.

[0183] HPLC parameters

[0184] Column: AAA C18 column (C18 reverse phase, 5μm, 4.6mm×150mm)

[0185] Temperature: 50°C

[0186] Mobile phase: A=H 2 O+0.1%FA B=Acetonitrile+0.1%FA

[0187] Injection volume: 20 μL

[0188] gradient:

[0189]

[0190] HPLC and MS parameters are typical and not limiting.

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Abstract

A method is described for mass spectrometric analysis of a sample comprising phenolic OH, such as a steroid comprising a phenolic OH, using a quaternary amino oxy Cookson (QAOC) reagent. The QAOC reagent can improve ionization and fragmentation properties of phenolic OH samples, which can thereby improve quantitation and identification. The method can include derivatizing the phenolic OH sample with the QAOC reagent to create an adduct and analyzing the adduct using mass spectrometry. Derivatization of the sample can be a one-step reaction where the QAOC reagent comprises an aminooxy MS tag or can be a multi-step reaction, where the adduct is formed by the reaction of carbonyl substituted PTAD based reagent and the sample followed by combination with an aminooxy MS tag. The sample can also be enriched prior to reacting it with the reagent. The method can also allow for multiplexing.

Description

[0001] Related US application [0002] This application claims priority from US Provisional Application Serial No. 61 / 588902, the entire teachings of which are incorporated herein by reference. technical field [0003] The present teaching relates to the field of mass spectrometry and labeling reagents suitable for mass spectrometry. Background technique [0004] Labeling chemistry is generally used to improve mass spectrometry signals. [0005] Use an aqueous olefinic reaction to derivatize using the reagent 4-phenyl-3H-1,2,4-triazole-3,5(4H)-dione (commonly known as PTAD or Cookson Reagent) Conjugation of tyrosine can provide conjugates that are particularly useful for the synthesis of bispecific antibodies (Hitoshi Ban, Jilia Gavrilyuk, Carlos F. Barbas, USA Journal of the Chemical Society (J. Am. Chem. Soc. 2010, 132, 1523-1525). PTAD has also been used to conjugate vitamin D species of compounds for mass spectrometric analysis (Kazutake Shimada, Tomoyuki Oe, Tatsuhi...

Claims

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Application Information

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IPC IPC(8): G01N30/72G01N33/50G01N33/487
CPCG01N2560/00G01N33/743G01N33/58G01N30/7233Y10T436/18Y10T436/200833Y10T436/203332
Inventor 萨巴卡·戴伊萨巴辛·普卡雅斯特哈
Owner DH TECH DEVMENT PTE