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 representative model of complex systems¹for studying the material basis of TCM formulas. Accurate, comprehensive, and reliable characterization of its chemical composition is a critical prerequisite for elucidating the pharmacodynamic material basis and improving quality control standards.

Ultra-High Performance Liquid Chromatography-Quadrupole-Electrostatic Field Orbitrap High-Resolution Mass Spectrometry (UPLC-QE-Orbitrap MS) has emerged as a core platform for analyzing chemical components in complex TCM systems, owing to its exceptional resolution, sensitivity, and mass accuracy. In terms of data acquisition strategies, Data-Dependent Acquisition (DDA) and Data-Independent Acquisition (DIA) are two mainstream modes^2,3,4(Figure 1). The DDA mode selectively triggers the fragmentation of high-abundance ions based on preset rules (e.g., precursor ion intensity thresholds, dynamic exclusion), yielding targeted high-quality MS/MS spectra that facilitate structural elucidation. However, it has limitations^5,6,7,8in covering low-abundance ions. In contrast, the DIA mode performs non-discriminatory fragmentation of all precursor ions within a specified mass-to-charge ratio range using continuous or variable windows. Theoretically, it captures all detectable ions, significantly enhancing the breadth and reproducibility of detection, particularly suitable for untargeted global analysis, though the resulting mixed MS/MS spectra are more complex to interpret^5,9.

Currently, while DDA and DIA techniques have been individually applied in TCM analysis, systematic, empirical comparative studies on core performance indicators, such as identification breadth (especially for low-abundance components), MS/MS spectrum quality, and detection sensitivity, within the same complex TCM formula system (e.g., Huaihua Powder) remain insufficient. Clarifying the applicable scenarios, advantages, and limitations of these two modes in complex TCM matrices is crucial for researchers to optimize strategy selection based on specific experimental objectives¹⁰.

Key parameters and limitations are summarized here to guide readers on the applicability of this protocol. The method is highly effective for profiling complex herbal matrices, with optimized parameters for a system containing flavonoids and alkaloids. The critical DIA variable window was set to 30 m/z after empirical testing; this parameter must be tuned for samples with different physicochemical properties. The method reliably detects low-abundance ions (peak area ~10⁵) but requires acquisition in separate positive and negative ionization modes to ensure data quality. A primary limitation is the dependence of DIA on spectral libraries and advanced deconvolution software for confident identifications, which influences computational resource requirements.

Accordingly, this study aims to conduct an in-depth and systematic comparison of the comprehensive performance of DDA and DIA scanning modes on the UPLC-QE-Orbitrap MS platform, using the classic formula Huaihua Powder as a case study. Optimized DDA and DIA scanning methods tailored for Huaihua Powder analysis will be developed. Data processing will be performed using Compound Discoverer software to evaluate and compare the two modes in the following dimensions: (1) total number of identified compounds; (2) quality of acquired MS/MS spectra; (3) reproducibility of peak areas and retention times of representative compounds; and 4) detection sensitivity for low-abundance active components. This research is expected to provide solid experimental evidence and data support for selecting high-resolution mass spectrometry scanning modes in studies of complex TCM systems, exploring the potential for complementary advantages of DDA and DIA to achieve a "balance between breadth and depth" and advance TCM modernization.

During the experiment, regarding the parameter setting of the segmented variable window in the DIA acquisition mode, we suggest setting it to 30 m/z. At the same time, the collection effect of positive and negative ions separately is better.

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 Rules for Preparation" in the 2020 edition of the Chinese Pharmacopoeia (Part IV). Take an appropriate amount of the original Platycladus orientalis leaves, remove impurities, place in a hot pan, and stir-fry over low heat (100 ºC) until yellow. Remove and cool.
3. Prepare the processed products of Zhike according to the method for "bran stir-fried Fructus Aurantii" in the 2020 edition of the Chinese Pharmacopoeia (Part I). Take an appropriate amount of raw Zhike material, remove impurities, heat to 100 °C, and sprinkle an appropriate amount of wheat bran at a ratio of 1:10 (materials to excipients).
   1. Once the smoke rises, add raw Zhike slices and stir-fry quickly. When the surface turns yellow or dark yellow, remove, sieve out the bran, cool, and set aside.
4. Prepare Huaihua Powder according to the 1:1:1:1 weight ratio prescription from Chinese Medical Formulology (New Century 3rd Edition). Accurately weigh 1 g of each herbal powder (sieved through a No. 5 sieve).
   1. Combine the powders in a mortar and mix according to the "trituration and mixing method" for traditional Chinese medicine powders: grind gently and consistently in one direction for 10 min.
   2. Consider the powder uniformly mixed when no visible streaks, agglomerates, or color variations are observed, resulting in a homogeneous appearance across the entire mixture. Prepare the final Huaihua Powder sample using this method.
5. Assess the mixing uniformity using a standardized visual inspection protocol. Examine the powder mixture under consistent lighting conditions against a white background.
   ​NOTE: Consider the mixture homogeneous only if it meets all of the following criteria: uniform color throughout with no visible streaks or patches of individual components; consistent texture without coarse or fine divergence; no obvious variation in color shade when sampled from multiple points; and no aggregated granular lumps detectable upon close inspection. Perform this assessment by two independent analysts to ensure consistency.
6. Weigh accurately 0.1 g of the well-mixed Huaihua Powder sample. Transfer the sample into a 10 mL volumetric flask. Add 8 mL of methanol.
7. Perform ultrasonic extraction for 60 min with a power of 500 W and a frequency of 40 kHz. Cool to room temperature. Make up the volume to the mark with methanol.
   1. Shake well to obtain a final concentration of 10 mg/mL Huaihua Powder extract. Filter through a 0.22 µm microporous membrane (PVDF). Transfer the filtrate into a sample vial for testing.
      ​NOTE: During the experiment, the organic waste liquid and other related toxic and harmful reagents were classified and labeled. And every 2 weeks, the staff of the Asset and Experimental Management Office of Chengdu University of Traditional Chinese Medicine transports it to a qualified institution for unified recycling and processing.

2. Detection conditions for UPLC-Q-Orbitrap HRMS

1. Chromatographic system parameter settings
   1. Use a C18 column (3.0 mm × 100 mm, 2.6 µm). Maintain the column temperature at 30 °C. Employ mobile phase A (0.1 % formic acid in water) and mobile phase B (acetonitrile).
   2. Program the gradient as follows: 0-10 min, 5%-20% B; 10-25 min, 20%-50% B; 25-40 min, 50%-95% B; 40-45 min, 95% B. Set the flow rate to 0.3 mL/min. Inject 5 µL of the sample.
2. Perform mass spectrometry study with the following parameter settings.
   ​NOTE: Ion source: Heated Electrospray Ionization (H-ESI); Scanning mode: In DDA acquisition mode, acquire both positive and negative ions simultaneously; in DIA acquisition mode, acquire positive and negative ions separately; Ion source parameters: Spray voltage +3.5 kV (positive ions)/-2.8 kV (negative ions); sheath airflow velocity 40 arb; auxiliary airflow velocity 10 arb; blowing gas velocity 1 arb; Ion transmission tube temperature: 320 °C; Evaporation temperature: 350 °C; S-Lens RF level: 55%.
   1. Construction and optimization of the Data-Dependent Acquisition (DDA) method
      1. For full MS Scan (Full MS), choose the following settings:
         ​NOTE: Resolution: 35,000; Scan range: 100-1000 m/z; Automatic Gain Control (AGC) Target: 3e6; Maximum injection time (Max IT): 100 ms.
      2. For Data-Dependent MS/MS Scan (dd-MS2), choose the following settings:
         1. For molecular ion selection, maintain the intensity threshold of 5e4. Exclude uncharged ions and isotope peaks (enable isotope exclusion). Apply dynamic exclusion with an exclusion time of 15 s and a mass tolerance of ±10 ppm to prevent repeated selection of high-intensity ions.
         2. For Fragmentation settings, maintain the following: Isolation window: 1.0 m/z; Fragmentation method: High-energy collision dissociation (HCD); Normalized collision energy (NCE): 20, 40, 60 V; Secondary resolution: 17,500; AGC Target: 1e5; Max IT: 50 ms; TopN strategy: Select the top 3 molecular ions with the highest intensity for fragmentation in each scan cycle.
   2. Construction and optimization of DIA Method
      1. For Full MS Scan, use the same parameters as in the DDA method.
      2. For Data-Independent MS/MS Scan (DIA-MS/MS), establish a segmented variable isolation window strategy.
      3. Define the target range as 100-1000 m/z. Divide windows based on ion abundance distribution across the entire m/z range using high-quality DDA spectra under optimized chromatographic conditions. Set variable windows to balance total ion abundance across segments, avoiding saturation or insufficiency.
      4. Optimize to 30 windows to balance coverage and cycle time.
         ​NOTE: Fragmentation settings - Fragmentation method: HCD; NCE: 20, 40, 60 V; Resolution: 17,500; AGC Target: 1e6; Max IT: Auto; Segmented variable window: 30 m/z.; Loop count: 30.
      5. Establish the inclusion list based on the central molecular weight of each isolation window segment, e.g., m/z 115, 145, 175, 205 ... 925, 955, 985.
   3. Data acquisition process
      1. System equilibration: Equilibrate the chromatographic column with the mobile phase for at least 5 min.
      2. Pre-experiment sample running: Prepare the Huaihua Powder test solution according to the preparation procedure. Inject 5 consecutive samples. Evaluate system stability (retention time RSD < 2 %, total ion current intensity RSD < 5 %).
      3. Blank running: Inject the methanol blank solution for background subtraction.
      4. Huaihua Powder sample running: Analyze each sample by injection in random order. Run all samples independently for each scanning mode (DDA, DIA).
   4. Data processing
      1. Compound identification
         1. Import the raw data acquired by mass spectrometry into the Compound Discoverer software. Establish an identification workflow for unknown components. Perform peak processing and peak alignment. Match measured MS/MS spectra with the mzCloud online MS/MS database and the mzVault local MS/MS database.
         2. Apply the following filtering parameters: peak area threshold 10⁵, mass deviation ± 5 ppm for both primary and secondary ions, and matching score ≥80. Compare filtered ions with compound information from databases, reference standards, and relevant literature to identify chemical components.
         3. Specific operation in the Compound Discoverer software: Create a new analysis process and select the Natural Product/Natural Product Unknown ID w Stats Online and Local Database Searchers workflow.
         4. Ensure inclusion of the Detect Compounds node for peak identification and extraction. Add the Search mzCloud and Search mzVault databases to the workflow.
         5. Add the raw data file and set filtering parameters (peak area threshold 10⁵, mass deviation ± 5 ppm for both primary and secondary ions). Click on run to execute the analysis.
      2. Comparative analysis of related indicators
         1. Identification quantity: Compare the total number of compounds identified in positive and negative ion modes for both DDA and DIA modes. Construct ion distribution maps for each mode.
         2. Reproducibility study: Compare retention time (RT) and peak area (AA) differences of representative compounds with high abundance (rutin, quercitrin), medium abundance (neohesperidin, naringin), and low abundance (psoralenol, trimethylcolchicinic acid) between DDA and DIA modes (n = 5).
         3. MS/MS spectrum quality: Evaluate the quality of MS/MS fragment ion spectra of pulegone, a key compound in Q-Marker, under DDA and DIA modes.
         4. Detection of low-abundance components: Compare the detection of MS/MS fragments of representative low-abundance compounds (carvone, adenosine) in DDA and DIA modes⁵.

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