Comparative Study of DDA and DIA Acquisition Modes on UPLC-Q-Orbitrap Mass Spectrometry Using Huaihua Powder

Summary

This study compares DDA and DIA modes (UPLC-QE-Orbitrap MS) for analyzing Huaihua Powder, a complex TCM. Optimized DDA and DIA methods were developed; compounds were identified via Compound Discoverer. Performance (compound identification, reproducibility, spectral quality, sensitivity) was evaluated, providing empirical guidance for TCM scanning mode selection.

Abstract

This study systematically evaluates the comparative performance of two mass spectrometry acquisition modes, data-dependent acquisition (DDA) and data-independent acquisition (DIA), coupled with ultra-high-performance liquid chromatography-quadrupole-orbitrap high-resolution mass spectrometry (UPLC-Q-Orbitrap HRMS) for comprehensive chemical profiling of complex traditional Chinese medicine (TCM) formulations. Huaihua Powder, a classical formulated prescription, was employed as a model system for empirical assessment. Optimized DDA and DIA acquisition methods were separately established: the DDA method incorporated a targeted precursor ion selection strategy with customized fragmentation parameters, while the DIA method employed a segmented variable window strategy to cover the target m/z range, performing unbiased fragmentation on all precursor ions. Compound identification was executed using Compound Discoverer software. The comparative evaluation specifically focused on the performance characteristics of the two acquisition modes, encompassing the number of identified compounds, reproducibility, MS/MS spectral quality, and detection sensitivity for low-abundance active constituents. Results demonstrated that the DDA mode yielded a higher total number of detected compounds, whereas the DIA mode generated a greater proportion of high-confidence identifications (10.63 % with spectral match scores >0.8). Notably, the DIA approach exhibited significantly superior reproducibility in retention time and peak area for six representative compounds, with rutin showing >3-fold difference in retention time RSD between the two acquisition modes. However, DDA produced cleaner MS/MS spectra with distinct fragment ions, whereas DIA spectra exhibited interference from contaminant ions. Concurrently, DIA effectively detected low-abundance active constituents whose ion chromatograms and MS/MS fragments could not be extracted in DDA mode. This study contributes critical experimental evidence and analytical datasets to inform the selection of high-resolution mass spectrometry acquisition modes for complex TCM formulation research. Subsequent researchers may integrate the complementary advantages of both approaches to achieve dual objectives in comprehensive compound characterization.

Introduction

Traditional Chinese medicine (TCM) formulas possess complex chemical systems with significant variations in component concentrations, particularly the presence of numerous low-abundance active ingredients, which pose severe challenges to analytical technologies. Huaihua Powder, a classical and renowned traditional Chinese medicine formula, is composed of four medicinal herbs: Sophora japonica, Platycladus orientalis leaves, Schizonepeta tenuifolia spikes, and Citrus aurantium peel. This formula contains a diverse array of bioactive substances, including flavonoids, alkaloids, and volatile oils, with significant variations in their concentrations. It serves as a repres....

Protocol

The reagents and the equipment used in this study are listed in the Table of Materials.

1. Preparation of the Huaihua Powder test solution

1. In accordance with the processing norms of "Stir-fried Sophora Flower" in the 2020 edition of the Chinese Pharmacopoeia (Part I), take an appropriate amount of the original Sophora flower material, remove impurities and branches, sieve out ash and debris, place in a hot pan, and stir-fry over low heat (100 °C) until dark yellow. Remove and cool.
2. Prepare the processed products of Platycladus orientalis leaves according to the "0213 General R....

Results

Overall identification of compounds

The total ion current chromatograms of Huaihua Powder under the two acquisition modes are shown in Figure 2, with minimal overall differences. In the DDA acquisition mode, a total of 14,958 compounds were identified in both positive and negative ion modes, while 9,489 compounds were identified in the DIA acquisition mode (Figu.......

Discussion

In this study, it is particularly worth mentioning that we found that in the DIA acquisition mode, the value setting of the segmented variable window should not be too large. We attempted to set it to 300mz and 100mz, but the final spectral effect was poor. After repeated exploration and attempts, we finally chose the acquisition window of 30mz as the most suitable. Therefore, the setting of the acquisition window has a crucial impact on the success or failure of the experiment.

At the same ti.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetonitrile	Thermo Fisher	/	Chromatographic purity
Compound Discoverer 3.3 software	Thermo Fisher	/	Mass spectrometry data processing and compound identification
fructus aurantii	Sichuan Neijiang	220709	The herbs in the prescription of Huaihua Powder
Platycladus orientalis leaves	Shandong Linyi	240619	The herbs in the prescription of Huaihua Powder
Schizonepeta spike	Jiangsu Taizhou	240601	The herbs in the prescription of Huaihua Powder
Sophora flower	Sichuan Guangan	241017	The herbs in the prescription of Huaihua Powder
Ultrasonic cleaning machine	Jietuo Ultrasonic Cleaning Equipment Co., LTD	240HT	Component extraction of Huaihua Powder
UPLC-Q-Orbitrap HRMS	Thermo Fisher	/	High-resolution mass spectrometer
Water	Watsons Company	/	Ultra-pure water