Investigation of In-Trap High-energy Collision Dissociation Methodology and Dissociation Conditions Using Dual-Pressure Linear Ion Trap Mass Spectrometry

Rationale: Dissociation is a critical analytical method in mass spectrometry research. Among various dissociation techniques, Higher-energy Collision Dissociation (HCD) stands out as a pivotal tool due to its ability to generate more low-mass fragment ions and directly perform dissociation during mass spectrometric detection. However, existing HCD implementations rely on specialized instrument configurations, such as collision cell structures or triple quadrupole mass spectrometers, which inevitably increase instrument development costs and technical complexity. To address this limitation, this study proposes an HCD method based on dual-pressure linear ion trap mass spectrometry that requires no additional hardware modifications. Methods: Instead, it achieves HCD solely by optimizing the instrument’s gas flow system and control parameters. By employing a nitrogen-helium gas mixture as the collision gas and testing with a reserpine standard sample, optimal dissociation conditions were obtained through adjustments to the gas mixture ratio and ion trap DC offset voltage settings. Results: This approach successfully realizes in-trap HCD directly within the dual-pressure linear ion trap, eliminating the need for a dedicated collision cell. Furthermore, it enables HCD-based MS3 functionality, a capability unavailable in traditional triple quadrupole systems. Conclusions: Validation experiments using veterinary drug residues fenoterol and ractopamine demonstrated the method’s universality by comparing its dissociation results with those from conventional ion trap Collision-Induced Dissociation (CID) and triple quadrupole method. The findings confirm that this strategy can be extended to other ion trap mass spectrometry platforms, offering broader applicability. Investigation of In-Trap High-energy Collision Dissociation Methodology and Dissociation Conditions Using Dual-Pressure Linear Ion Trap Mass Spectrometry Heyi Xu a | You Jiang b | Jie Xie b | Li Yao c | Xiang Fang b | Meiying Liu b | Ziyu Qu b | Guang Yang a,* | Di Zhang b,* a College of Instrumentation Electrical Engineering, Jilin University, Changchun 130061, Jilin, PR China | b Technology Innovation Center of Mass Spectrometry for State Market Regulation, Center for Advanced Measurement Science, National Institute of Metrology, Beijing 100029, PR China | c College of Earth Sciences, Jilin University, Changchun 130061, Jilin, PR China Correspondence: Guang Yang ([email protected]) | Di Zhang ([email protected]) Funding: This work was supported by the National Key R&D Program of China (No. 2022YFF0705001)，the NIM research foundation (No. AKYZZ2122) and the SAMR Scientific and Technological Programs (No. S2024MK0553).

high-energy collision dissociation | double pressure linear ion trap mass spectrometer | collision gas | multistage mass spectrometry analysis | veterinary drugs detection

Rationale: Dissociation is a critical analytical method in mass spectrometry research. Among various dissociation techniques, Higher-energy Collision Dissociation (HCD) stands out as a pivotal tool due to its ability to generate more low-mass fragment ions and directly perform dissociation during mass spectrometric detection. However, existing HCD implementations rely on specialized instrument configurations, such as collision cell structures or triple quadrupole mass spectrometers, which inevitably increase instrument development costs and technical complexity. To address this limitation, this study proposes an HCD method based on dual-pressure linear ion trap mass spectrometry that requires no additional hardware modifications.

Instead, it achieves HCD solely by optimizing the instrument’s gas flow system and control parameters. By employing a nitrogen-helium gas mixture as the collision gas and testing with a reserpine standard sample, optimal dissociation conditions were obtained through adjustments to the gas mixture ratio and ion trap DC offset voltage settings.

This approach successfully realizes in-trap HCD directly within the dual-pressure linear ion trap, eliminating the need for a dedicated collision cell. Furthermore, it enables HCD-based MS3 functionality, a capability unavailable in traditional triple quadrupole systems.

Validation experiments using veterinary drug residues fenoterol and ractopamine demonstrated the method’s universality by comparing its dissociation results with those from conventional ion trap Collision-Induced Dissociation (CID) and triple quadrupole method. The findings confirm that this strategy can be extended to other ion trap mass spectrometry platforms, offering broader applicability. 1 | Introduction The Dissociation techniques are commonly employed to fragment molecules for structural analysis, where the molecular structure is elucidated through the analysis of fragment ions in mass spectrometry research. These dissociation techniques require substantial energy to break apart molecules. Dissociation techniques require substantial energy to break apart molecules and are broadly categorized into two groups: those requiring background gases, such as conventional Collision-Induced Dissociation (CID) and its derivatives, and those not requiring background gases, such as Ultraviolet Photodissociation (UVPD) and other methods utilizing lasers or alternative technologies [1-3] . Additional background gas-dependent dissociation methods include pulsed-Q dissociation (PQD), dipolar excitation dissociation, broadband dissociation, multi-generational collision-induced dissociation, red-shifted off-resonance large-amplitude excitation (RSORLAE), dynamic collision-induced dissociation (DCID), and dipolar DC collisional activation (DDC). Conversely, background gas-independent methods encompass surface-induced dissociation (SID), infrared multiphoton dissociation (IRMPD), and electron-transfer dissociation (ETD), each applied in specific scenarios based on their unique characteristics [1] . Among these, UVPD has been reported to exhibit rapid dissociation kinetics and generate rich fragment ion profiles, making it a cutting-edge technique in current research. However, its widespread adoption in mass spectrometry studies remains limited due to the high development costs associated with integrating laser systems [4-5] . Conventional CID is the most widely used dissociation method in mass spectrometry these days. Its fundamental principle involves accelerating precursor ions to high kinetic energies, after which they undergo collisions with neutral gas molecules (e.g., helium, nitrogen) in the mass analyzer. During these collisions, a portion of the kinetic energy is converted into internal energy, causing specific bonds within the molecular structure to break and generate fragment ions [6] . This method is readily implemented on standard mass spectrometers, offering simplicity and meeting basic analytical requirements [7-8] . However, it exhibits limitations in detecting low-mass fragment ions, particularly yielding fewer ions with mass-to-charge ratios (m/z) below 200. This shortfall compromises the accuracy of compositional analysis for target molecules. To address such issues, another CID mode employs sequential collision technology, a representative example of which is the triple quadrupole mass spectrometer [9] . The instrument features a triple quadrupole configuration, in which Q2 functions as a collision cell for CID, while Q1 serves as the mass selector and Q3 as the mass analyzer. This design promotes more complete ion fragmentation and generates additional low-mass fragment ions. However, such spectrometers lack ion trap or other ion storage devices, making them incapable of performing MSn (n≥3) multistage dissociation, which limits their applicability in certain scenarios. Consequently, a CID-derived fragmentation method called High-energy Collision Dissociation (HCD) was developed. This technique incorporates a high-energy collision cell into the mass spectrometer. During detection, ions are directed into this collision cell where a higher DC voltage potential difference is applied. This generates elevated energy levels that induce more extensive fragmentation compared to conventional CID, thereby producing richer low-mass fragment ions and enhanced mass spectral information [10-11] . Currently, Thermo Scientific’s Orbitrap series represents the primary mass spectrometry platform implementing HCD in research applications. The company’s recently developed Stellar mass spectrometer further advances detection capabilities, enabling superior dissociation analysis and standing as the state-of-the-art HCD-enabled instrument. Although HCD technology has reached relative maturity, room for improvement remains. The requirement of a dedicated collision cell necessitates additional instrument components [12], which compromises structural compactness, increases R&D costs and complexity, and conflicts with the current industry trend toward miniaturized, portable, and field-deployable detection systems. Based on the aforementioned requirements, this study proposes a HCD method utilizing a dual-pressure linear ion trap. This approach repurposes the linear ion trap, conventionally employed for mass analysis in mass spectrometers, as the high-energy collision cell for dissociation [13] . By elevating the DC offset voltage of the ion trap to create a potential gradient, ions simultaneously undergo dissociation and detection directly within the ion trap during routine scanning. However, experimental results revealed that the dissociation efficiency of this ion trap-based collision cell was lower compared to conventional methods [14-16] . To address this, reserpine standard samples were utilized to investigate strategies for enhancing dissociation efficiency and increasing the absolute intensity of characteristic fragment ions under this configuration. Key parameters optimized included collision gas composition, flow rate, and ion trap DC offset voltage, with experimental comparisons identifying the optimal dissociation conditions. Furthermore, this method could achieve HCD-based MS3 functionality [17-18], which is unavailable in triple quadrupole mass spectrometers. Specifically, the SWIFT (Stored Waveform Inverse Fourier Transform) capability of the ion trap was employed to isolate first-generation fragment ions from reserpine. Subsequent adjustment of the DC offset voltage to establish a high potential gradient induced secondary fragmentation, yielding second-generation fragment ions. Additionally, this approach was applied to veterinary drug residues such as fenoterol and ractopamine. HCD successfully fragmented dehydration peaks of precursor ions that CID failed to dissociate effectively, while generating higher abundances of low-mass terminal fragment ions. Comparative studies with ion trap CID and triple quadrupole mass spectrometry confirmed the universal applicability of this method. This method utilizes an ion trap as the collision cell, eliminating the need for a dedicated high-energy collision cell for dissociation or complex structural modifications to the instrument. By employing a mixed-gas approach, it ensures dissociation efficiency and resolution, making it readily applicable to other types of mass spectrometers equipped with ion trap structures. 2 | Experimental 2.1 | Materials and Reagents The study utilized standard solid samples of reserpine (m/z=609), fenoterol (m/z=304), and ractopamine (m/z=302) as experimental method development and test samples. Other reagents included anhydrous methanol, high-purity water, high-purity helium and nitrogen (used as collision gas for the mass analyzer), and standard nitrogen (used as sheath and auxiliary gas for the ion source). Reserpine was dissolved in a methanol-water mixture (1:1, v/v) to prepare a 100 ppb solution. Fenoterol and ractopamine were individually dissolved in methanol to prepare 100 ppb solutions. All solutions were introduced into the system by syringe pump infusion. 2.2 | Instrumentation The experimental platform employed in this study is a quadrupole-dual-pressure linear ion trap tandem mass spectrometry system independently developed by the National Institute of Metrology (NIM), China. As illustrated in the Figure 1, its components divided into electrospray ionization source composed of a 0.1 mm quartz capillary and a 0.25 mm metal capillary, the maximum operating voltage is 6 kV, the maximum temperature is 500°C and the maximum flow rate is 1 mL/min; multi-stage vacuum system including first stage 1-3 Torr, second stage 0.05-0.1 Torr, third stage 5×10 -4 -1.2×10 -5 Torr and fourth stage 5.0×10 -6 -1.0×10 -5 Torr; the ion transport system including RF ion guide (Q-JET) with a frequency of 1-1.5 MHz, a bent quadrupole with RF frequencies of 2-3 MHz; the mass analysis system comprises an analytical quadrupole and dual linear ion traps. The quadrupole and ion trap 1 operate at a pressure range of 1×10 −3 -5×10 −3 Torr, while ion trap 2 maintains a pressure of 1×10 −4 -1×10 −3 Torr.; the detector system with a photomultiplier and dynode operating at -1 to 1.5 kV. The operational timing sequence for HCD’s ion detection in this instrument is depicted in Figure 2 . Samples are introduced into the nano-electrospray ionization source through the injection system. Ions generated in the source are efficiently captured by a contamination-resistant atmospheric pressure interface. Under electric fields, ions are transported through a multi-stage ion guidance system (comprising ion guides and lenses) into high-vacuum regions. The mass analyzer, consisting of a quadrupole and two linear ion traps in series, operates as follows: ions are selected by the quadrupole and transferred to ion trap 1, where they are stored through collisions with buffer gas and electric fields; after dissociation in ion trap 1, ions enter ion trap 2 for analysis and detection [19-21] . Compared to single-ion-trap systems, the dual-pressure design enhances trapping and fragmentation efficiency in the high-pressure trap (ion trap 1) while enabling faster scanning speeds and improved mass resolution in the low-pressure trap (ion trap 2) [22-23] . Collisional dissociation typically employs helium or nitrogen as the collision gas. Studies have shown that nitrogen enhances dissociation efficiency but compromises mass resolution, while helium maintains resolution but yields lower dissociation efficiency [24-25] . To address this trade-off, this study investigates the effects of helium-nitrogen gas mixtures on both dissociation efficiency and mass resolution. A novel platform modification was implemented (as illustrated in the Figure 1 ) to enable flexible gas composition adjustment. The conventional single gas line configuration was redesigned into a dual parallel gas line system. Each line features independent valves and flow controllers (Alicat DS series, maximum flow rate 5 sccm) to regulate gas switching and flow rates. This allows simultaneous introduction of high-purity helium and nitrogen into the ion trap as a mixed collision gas and isolation of either gas for single-component experiments. This modification facilitates systematic exploration of collision gas composition and concentration, balancing dissociation efficiency with spectral resolution. FIGURE 1 | Instrumentation modification for gas mixture control. FIGURE 2 | The operational timing sequence for HCD’s ion detection. 3 | Results and Discussion 3.1 | Investigation of Gas Content and Ion Trap DC Offset Voltage This study optimized two key parameters: collision gas composition and pressure and the DC offset voltage of ion trap 1. Using reserpine standard samples as the test subject, optimization and adjustment of in-trap HCD were conducted in the dual-pressure linear ion trap mass spectrometer. The linear ion trap has a larger ion capacity compared to other ion traps, which is more conducive to ion storage [26-28] . As illustrated in the Figure 3 [29], the linear ion trap structure comprises three sequential sections (front, center, and back), each requiring distinct DC offset voltages for ion manipulation to ensure effective ion storage and detection. Since the target ions for dissociation predominantly occupy the spatially largest center section, increasing the DC offset voltage in this region theoretically enhances the electric potential gradient, thereby promoting dissociation efficiency. Experimental iterations confirmed that adjusting the front and back sections’ voltages had negligible effects on dissociation outcomes. Consequently, this research focused exclusively on optimizing the middle segment’s DC offset voltage. The proposed methodology involves first optimizing the collision gas composition and flow rate. Since excessively low or high pressure within the ion trap compromises stable mass spectral signals, the total gas flow must be adjusted within a range that maintains appropriate pressure. However, as pressure cannot be directly regulated in these experiments, it is indirectly controlled by modulating the flow rates of two gases. Tests were conducted under three gas conditions: pure helium, pure nitrogen, and a helium-nitrogen mixture, with flow rates fine-tuned to ensure stable signal acquisition. Subsequently, the DC offset voltage of the center section in ion trap 1 was systematically tuned. The dissociation efficacy was evaluated using the low-mass diagnostic fragment ion m/z=195, generated from the precursor reserpine ion (m/z=609), as the primary metric. This fragment exhibits low abundance under conventional CID conditions. Dissociation performance was quantified via the absolute signal intensity of m/z=195 and dissociation efficiency, calculated as the ratio of the fragment ion’s intensity to the precursor ion’s intensity. Total dissociation efficiency, accounting for all fragment ions, was also computed for reference. Notably, under pure nitrogen or nitrogen-rich conditions, mass resolution deteriorates significantly, particularly for high m/z ions such as reserpine (m/z=609). To mitigate this, the full width at half maximum (FWHM) of both the precursor (m/z=609) and diagnostic fragment (m/z=195) was monitored to identify operating ranges where resolution remained acceptable. Optimization proceeded only within these validated ranges. Ultimately, the synergistic combination of gas condition and ion trap voltage yielded optimal dissociation conditions tailored for reserpine standard samples. FIGURE 3 | Geometric Structure of Linear Ion Trap [29] . 3.1.1 | Pure Helium Gas Conditions In the study of ion trap HCD, pure helium was used as the collision gas without nitrogen addition. The DC offset voltages of the three segments of Ion Trap 1 were set to -20 V, -30 V, and 25 V, respectively. The relationships between helium flow rate and intensity of the m/z=195 fragment after dissociation and reserpine (m/z=609) before dissociation and the dissociation efficiency of m/z=195 relative to the parent ion reserpine is illustrated in the Figure 4 . The intensity of m/z=195 peaks at 4.5 sccm, while the dissociation efficiency reaches its maximum at a helium flow rate of 4 sccm. Considering that excessive helium content (as previously mentioned) may lead to overpressure during subsequent nitrogen introduction, thereby compromising detection performance, helium flow rate of 4 sccm was selected. The corresponding mass spectrum under this condition is illustrated in the Figure 5 . It should be noted that when the helium flow rate exceeds 4.5 sccm, the trap pressure surpasses the threshold, resulting in reduced detection sensitivity. Both the absolute signal intensities of the m/z=195 fragment and reserpine (m/z=609) exhibit significant declines under such conditions. Therefore, these parameters fall outside conventional experimental settings, and calculated dissociation efficiencies under these circumstances should not be considered representative of general trends. (A) (B) FIGURE 4 | The relationships between helium flow rate and (A) intensity of the m/z=195 fragment and reserpine (m/z=609), (B) dissociation efficiency of m/z=195 when helium only. FIGURE 5 | HCD mass spectrum of reserpine at helium flow rate of 4 sccm. 3.1.2 | Pure Nitrogen Gas Conditions In This time pure nitrogen was used as the collision gas without helium addition in the study of ion trap HCD. The DC offset voltages of the three segments of ion trap 1 were set to -20 V, -30 V, and 25 V, respectively. Since increasing the nitrogen flow rate reduces the mass resolution of spectral peaks—particularly for the pre-dissociation reserpine (m/z=609) with a larger mass-to-charge ratio, it is necessary to monitor the full width at half maximum (FWHM) of m/z=609 and 195 during adjustments to the nitrogen flow rate. This ensures the selection of a flow rate range that does not significantly degrade mass resolution before proceeding with investigations into dissociation parameters. The relationships between nitrogen flow rate and FWHM, intensity of the m/z=195 fragment after dissociation and reserpine (m/z=609) before dissociation and dissociation efficiency of m/z=195 fragment relative to the parent ion and reserpine (m/z=609) are illustrated in the Figure 6 . When the nitrogen flow rate exceeds 0.3 sccm, reserpine’s FWHM shows a marked increase, accompanied by significant degradation of mass resolution. Thus, nitrogen flow rates below 0.3 sccm were selected for dissociation parameter optimization. At 0.3 sccm, the m/z=195 fragment achieves maximum absolute signal intensity. Although dissociation efficiency continues to improve with higher flow rates, such conditions fall outside the acceptable range for maintaining mass resolution. Consequently, 0.3 sccm was identified as the optimal nitrogen flow rate, with corresponding mass spectra illustrated in the Figure 7 . (A) (B) (C) FIGURE 6 | The relationships between nitrogen flow rate and (A) FWHM, (B) intensity of the m/z=195 fragment and reserpine (m/z=609), (C) dissociation efficiency of m/z=195 when nitrogen only. FIGURE 7 | HCD mass spectrum of reserpine at nitrogen flow rate of 0.3 sccm. 3.1.3 | Mixed Gas Conditions Current Investigating the optimal configuration of mixed gases requires incrementally introducing a secondary gas into a primary single-component gas to achieve the most suitable gas composition. Given that nitrogen has a higher molecular weight than helium, it dominates the dissociation effect at equivalent flow rates. Consequently, the addition of helium to a high-flow nitrogen environment exerts minimal influence on dissociation. Experimental validation confirmed that dissociation outcomes using nitrogen-dominated mixtures with trace helium as the collision gas show no significant deviation from those using pure nitrogen. Furthermore, as demonstrated in prior studies, elevated nitrogen flow rates markedly degrade mass resolution, adversely impacting detection performance. Under practical constraints, the recommended methodology for mixed-gas collision systems involves progressively blending small amounts of nitrogen into a high-flow helium-dominated baseline. This approach allows systematic exploration of optimal dissociation conditions while maintaining acceptable mass resolution. Building upon this foundation, dissociation studies were conducted using a mixed collision gas system by introducing nitrogen into helium flow rate of 4 sccm, as previously determined. The relationships between nitrogen flow rate and intensity and dissociation efficiency of m/z=195 fragment relative to the parent ion and reserpine (m/z=609) are illustrated in the Figure 8 . Overall, a declining trend in absolute signal intensity was observed, primarily attributed to the elevated pressure caused by helium-dominated gas mixtures approaching the detection threshold as nitrogen was added. Consequently, subsequent investigations were designed to vary helium flow rates under fixed nitrogen conditions. At the nitrogen flow rate of approximately 0.03 sccm, the highest absolute signal intensity and dissociation efficiency for the m/z=195 fragment were achieved. (A) (B) FIGURE 8. The relationships between nitrogen flow rate and (A) intensity of the m/z=195 fragment and reserpine (m/z=609), (B) dissociation efficiency of m/z=195 when mixed with helium flow rate of 4 sccm. With the nitrogen flow rate fixed at 0.03 sccm, the helium flow rate was incrementally adjusted. The relationships between helium flow rate and (A) intensity of the m/z=195 fragment and reserpine (m/z=609) and (B) dissociation efficiency of the m/z=195 fragment relative to the parent ion reserpine are illustrated in the Figure 9 . Helium flow rate of 3.5 sccm yielded the highest absolute signal intensity and dissociation efficiency, with the corresponding mass spectrum illustrated in the Figure 10 . (A) (B) FIGURE 9 | The relationships between helium flow rate and (A) intensity of the m/z=195 fragment and reserpine (m/z=609), (B) dissociation efficiency of m/z=195 when mixed with nitrogen flow rate of 0.03 sccm. FIGURE 10 | HCD mass spectrum of reserpine at helium flow rate of 3.5 sccm and nitrogen flow rate of 0.03 sccm. The aforementioned studies established a set of gas compositions. However, since helium flow rates were maintained above 1 sccm to ensure stable detection signals in the mass spectrometer, further validation is required to determine whether better dissociation conditions could be achieved by reducing helium flow below 1 sccm, which would allow a broader nitrogen flow adjustment range. Under these conditions, while helium remains the primary component of the collision gas, the increased proportion of nitrogen necessitates careful consideration of its impact on mass resolution. With helium fixed at 0.8 sccm, the relationships between nitrogen flow rate and FWHM, intensity of the m/z=195 fragment and reserpine (m/z=609) and dissociation efficiency of the m/z=195 fragment relative to the parent ion are illustrated in the Figure 11 . Reserpine’s FWHM exhibited a marked increase beyond 0.1 sccm nitrogen flow, while absolute signal intensity peaked at this point. Although dissociation efficiency continued to improve with higher nitrogen flow rates, both mass resolution and absolute signal intensity progressively declined. Consequently, nitrogen flow should not exceed levels that compromise resolution. The optimal nitrogen flow rate was determined to be 0.1 sccm, balancing maximal absolute signal intensity with acceptable mass resolution. (A) (B) (C) FIGURE 11 | The relationships between nitrogen flow rate and (A) FWHM, (B) intensity of the m/z=195 fragment and reserpine (m/z=609), (C) dissociation efficiency of m/z=195 when mixed with helium flow rate of 4 sccm. With nitrogen flow fixed at the previously determined optimal 0.1 sccm, helium flow was incrementally adjusted. Although the initial objective focused on helium flows below 1 sccm, the low nitrogen flow rate (0.1 sccm) allowed helium to be increased up to 3 sccm while maintaining stable detection signals. Consequently, the study evaluated helium flow variations across a 0-3 sccm range. The relationships between helium flow rate and intensity of the m/z=195 fragment and reserpine (m/z=609) and dissociation efficiency of the m/z=195 fragment relative to the parent ion reserpine are illustrated in the Figure 12 . Both absolute signal intensity and dissociation efficiency simultaneously peaked at helium flow rate of 1 sccm, with the corresponding mass spectrum illustrated in the Figure 13 . (A) (B) FIGURE 12 | The relationships between helium flow rate and (A) intensity of the m/z=195 fragment and reserpine (m/z=609), (B) dissociation efficiency of m/z=195 when mixed with nitrogen flow rate of 0.1 sccm. FIGURE 13 | HCD mass spectrum of reserpine at helium flow rate of 1 sccm and nitrogen flow rate of 0.1 sccm. 3.1.4 | Ion Trap DC Offset Voltage Conditions Following the establishment of optimal gas conditions, further adjustments were made to the DC offset voltage of the ion trap 1 center section. Under pure helium flow rate of 4 sccm, the relationship between the DC offset voltage value and the intensity and dissociation efficiency of the m/z=195 fragment is illustrated in the Figure 14. Since the gas flow rate was fixed, the intensity of the parent ion reserpine (m/z=609) remained largely constant prior to dissociation, allowing focused analysis on the behavior of the m/z=195 fragment. Notably, as the detected ions are positively charged, the DC offset voltage of ion trap 1 center section was set to negative polarity to ensure efficient ion transmission and storage. For clarity in visualizing voltage trends, the polarity of the voltage values was inverted during adjustment (e.g., -120 V reported as 120 V), a convention maintained throughout subsequent analyses. As shown in the Figure 14, DC offset voltage of -120 V (nominally reported as 120 V) in the center section of ion trap 1 yielded the highest absolute signal intensity of 139.8 mV and dissociation efficiency of 17.56% for the m/z=195 fragment, representing the optimal dissociation performance under these conditions. FIGURE 14 | The relationships between intensity, dissociation efficiency of the m/z=195 fragment and the dc offset voltage of ion trap center section when helium flow rate of 4 sccm only. In Under pure nitrogen flow rate of 0.3 sccm, the DC offset voltage of ion trap 1 center section was adjusted. The relationships between offset voltage values and intensity of m/z=195, as well as dissociation efficiency, were illustrated in the Figure 15. The optimal dissociation effect was achieved at -60 V, yielding intensity of 97.5 mV for m/z=195 and a fragment dissociation efficiency of 21.72%. FIGURE 15 | The relationships between intensity, dissociation efficiency of the m/z=195 fragment and the dc offset voltage of ion trap center section when nitrogen flow rate of 0.3 sccm only. Under helium flow rate of 3.5 sccm and nitrogen flow rate of 0.03 sccm, the DC offset voltage of ion trap 1 center section was adjusted. The relationships between offset voltage values and intensity of m/z=195, as well as dissociation efficiency, were illustrated in the Figure 16. The optimal dissociation effect was achieved at -100 V, yielding intensity of 157.4 mV for m/z=195 and a fragment dissociation efficiency of 23.56%. FIGURE 16 | The relationships between intensity, dissociation efficiency of the m/z=195 fragment and the dc offset voltage of ion trap center section when helium flow rate of 3.5 sccm and nitrogen flow rate of 0.03 sccm. Under helium flow rate of 1 sccm and nitrogen flow rate of 0.1 sccm, the DC offset voltage of ion trap 1 center section was adjusted. The relationships between offset voltage values and intensity of m/z=195, as well as dissociation efficiency, were illustrated in the Figure 17. The optimal dissociation effect was achieved at -65 V, yielding intensity of 212.5 mV for m/z=195 and a fragment dissociation efficiency of 26.93%. FIGURE 17 | The relationships between intensity, dissociation efficiency of the m/z=195 fragment and the dc offset voltage of ion trap center section when helium flow rate of 1 sccm and nitrogen flow rate of 0.1 sccm. The main research findings regarding the gas and the DC offset voltage conditions of ion trap 1 center section are summarized in the Table 1, which can be categorized into three groups: pure helium, pure nitrogen, and mixed gas. A comprehensive comparison of the parameters clearly demonstrates the advantages of using mixed gas. Under the mixed gas conditions, higher dissociation efficiency was achieved with helium at 1 sccm, nitrogen at 0.1 sccm, and the DC offset voltage -65 V of ion trap 1 center section. Compared to the conditions of helium at 3.5 sccm, nitrogen at 0.03 sccm, and the DC offset voltage -100 V. Additionally, the required the DC offset voltage -65 V is lower in magnitude than -100 V, which is more favorable for the stable operation of the instrument. Furthermore, analysis of the optimal dissociation conditions derived from the reserpine standard samples revealed that gas parameters predominantly affect instrument performance and can thus be standardized as fixed conditions applicable to dissociation studies of diverse analytes. However, due to structural variations among different compounds, the required dissociation energy varies significantly. Consequently, the ion trap DC offset voltage must be retuned according to the specific analyte type, following a process analogous to the methodology detailed in the aforementioned study. Therefore, in subsequent sections, the investigation of ion trap DC offset voltage will not reiterate procedural specifics; only the optimal dissociation voltage, dissociation efficiency, and other critical parameters will be explicitly reported. TABLE 1 | Relationship between gas conditions, ion trap center section DC offset voltage conditions, and dissociation parameters. | Gas Condition | DC Offset Voltage of Ion Trap 1 Center Section(V) | Intensity of m/z=609 Before Dissociation (mV) | Intensity of m/z=195(mV) | Total Dissociation Intensity(mV) | Dissociation Efficiency of m/z=195 | Total Dissociation Efficiency | FWHM of m/z=195 | FWHM of m/z=609 | | He 4 sccm | -120 | 796.0 | 139.8 | 297.3 | 17.56% | 37.35% | 0.2 | 0.27 | | N2 0.3 sccm | -60 | 449.0 | 97.5 | 220.0 | 21.72% | 48.99% | 0.29 | 0.40 | | He 3.5 sccm N2 0.03 sccm | -100 | 668.0 | 157.4 | 456.5 | 23.56% | 68.34% | 0.23 | 0.30 | | He 1 sccm N2 0.1 sccm | -65 | 789.0 | 212.5 | 520.3 | 26.93% | 65.95% | 0.24 | 0.32 | 3.2 | HCD-based MS³ Analysis HCD-based MS 3 Analysis. Under the optimized conditions mentioned above, MS 2 dissociation of reserpine was achieved using a dual-pressure linear ion trap. Building on this, leveraging the ion trap functionality of the dual-pressure linear ion trap mass spectrometer, further research on HCD-based MS 3 can be conducted. Utilizing the SWIFT (Stored Waveform Inverse Fourier Transform) waveform filtering capability of the ion trap, the primary dissociation fragment of reserpine (m/z=609) generated from the previously studied MS 2 process, specifically the m/z=448 fragment was isolated, as illustrated in the figure. This primary dissociation fragment was first transferred to the front section of the ion trap for storage. Subsequently, by adjusting the DC offset voltages of the three segments of the ion trap, the primary fragment was moved to the middle section of the ion trap for further dissociation, yielding secondary fragments. As shown in the Figure 18, the resulting fragments at m/z=195 and 236 were confirmed to originate from the secondary dissociation of the m/z=448 fragment. This demonstrates that the dual-ion-trap-based HCD approach enables MS 3 functionality with ion trap characteristics. The corresponding MS 3 mass spectra are presented in the figures. On this basis, the relationship between the center section’s DC offset voltage of the ion trap (a key factor influencing dissociation) and the MS 3 dissociation efficiency of the characteristic fragment at m/z=195 was investigated. Following the same experimental approach as described previously, the highest dissociation efficiency of 46.6% was achieved at a DC offset voltage of -60 V. This study confirms that the in-trap direct HCD method proposed here enables MS 3 multi-stage dissociation with inherent ion trap characteristics, thereby expanding its applicability. Furthermore, it demonstrates the analytical advantages of integrating ion trap functionality (e.g., voltage control and fragmentation) with HCD as a collision cell, eliminating the need for external modules enhancing sensitivity, cost-effectiveness, and compatibility with advanced workflows like MS 3 which is capability unattainable in triple quadrupole systems. FIGURE 18 | HCD-based MS 2 and MS 3 Dissociation Mass Spectra of Reserpine (m/z = 609). 3.3 | HCD-Based Detection of Veterinary Drug Residues β-adrenergic receptor agonists are a class of drugs that can bind to and activate adrenergic receptors, producing adrenaline-like effects. Due to their ability to significantly promote animal growth, increase lean meat yield, reduce fat deposition, and improve feed conversion efficiency, they have been widely used in animal production. However, studies have found that the use of these drugs can easily accumulate in animal products, posing potential hazards to human health. Therefore, qualitative detection of veterinary drug residues has become a critical requirement in the field of food safety [30] . Since ion trap CID fails to effectively dissociate the dehydration peaks of such substances and obtain characteristic low-mass fragments for compositional identification, triple quadrupole mass spectrometry is typically employed for their dissociation analysis. However, the HCD-based in-trap dissociation method proposed in this study retains the specificity of conventional HCD, while also enabling dissociation and qualitative analysis of these compounds. The dissociation analysis was conducted using two β-adrenergic receptor agonist compounds: fenoterol (m/z=304) and ractopamine (m/z=302). Dissociation studies on the fenoterol sample (m/z=304) were performed using ion trap HCD, ion trap CID, and the SCIEX AB 6500+ triple quadrupole mass spectrometer. The dissociation results are shown in Figure 19 . Since the signal intensity unit for the triple quadrupole mass spectrometer was fixed as counts per second (cps), the signal intensity units for ion trap dissociation spectra were also switched to cps via control software for consistency, with the same approach applied hereafter. Moreover, the use of a nitrogen-helium mixed gas as the collision gas in the aforementioned experimental ion trap serves to preserve resolution, whereas the resolution of the triple quadrupole mass spectrometer is not compromised by nitrogen. Therefore, pure nitrogen can be directly employed as the collision gas [9] . Comparative analysis revealed that after ion trap HCD treatment, the parent ion and dehydration peak (m/z=286) were nearly completely dissociated, yielding low-mass structural characteristic fragments such as m/z=107, m/z=135, and m/z=152, enabling qualitative identification of fenoterol. The highest dissociation efficiency was achieved at a mid-trap DC offset voltage of -50 V, as tabulated. In contrast, although ion trap CID dissociated the main components, significant residual dehydration peaks (m/z=286) persisted, with minimal low-mass dissociation fragments observed. The triple quadrupole mass spectrometer produced characteristic ions similar to those from ion trap HCD for fenoterol, with the dissociation efficiency of the m/z=107 fragment closely matching that of the ion trap HCD method, as detailed in Table 2 . (A) (B) (C) FIGURE 19 | Dissociation mass spectra of fenoterol (m/z 304) obtained by (A) ion trap HCD, (B) ion trap CID, (C) triple quadrupole mass spectrometry. TABLE 2 | Comparison of the dissociation efficiencies of fragment ions of fenoterol following HCD in ion trap and dissociation in triple quadrupole mass spectrometer. | Type of Fragment Ions | m/z=107 | m/z=135 | m/z=152 | | Dissociation Efficiencies of Ion Trap HCD | 18.56% | 37.39% | 7.33% | | Dissociation Efficiencies of Triple Quadrupole Mass Spectrometer | 17.14% | 50.90% | 3.71% | Similarly, dissociation studies on the fenoterol sample (m/z=304) were conducted using ion trap HCD, ion trap CID, and the SCIEX AB 6500+ triple quadrupole mass spectrometer, with dissociation results shown in Figure 20 . Comparative analysis revealed that after ion trap HCD, the parent ion and dehydration peak (m/z=284) were nearly completely dissociated, yielding structural characteristic fragments at m/z=164 and m/z=107 (identical to fenoterol), enabling qualitative analysis of ractopamine. The highest dissociation efficiency was achieved at -55 V mid-trap DC offset voltage in the ion trap, as tabulated. While ion trap CID successfully removed the main components, residual dehydration peaks (m/z=284) remained abundant, with limited low-mass dissociation fragments observed. In contrast, the triple quadrupole mass spectrometer demonstrated more complete dissociation of ractopamine, generating a greater variety of fragment ions compared to ion trap HCD. However, ion trap HCD still produced identifiable characteristic fragments such as m/z=107 and m/z=164 for structural elucidation. Notably, the dissociation efficiency of the m/z=107 fragment was comparable between the triple quadrupole and ion trap HCD methods, as detailed in Table 3 . This applied research confirms that the ion trap-based HCD method retains the specificity of conventional HCD detection, providing an effective approach for qualitative analysis of veterinary drug residues. (A) (B) (C) FIGURE 20 | Dissociation mass spectra of ractopamine (m/z 302) obtained by (A) ion trap HCD, (B) ion trap CID, (C) triple quadrupole mass spectrometry. TABLE 3 | Comparison of the dissociation efficiencies of fragment ions of ractopamine following HCD in ion trap and dissociation in triple quadrupole mass spectrometer. | Type of Fragment Ions | m/z=107 | m/z=121 | m/z=136 | m/z=164 | | Dissociation Efficiencies of Ion Trap HCD | 15.25% | / | / | 45.36% | | Dissociation Efficiencies of Triple Quadrupole Mass Spectrometer | 17.44% | 20.32% | 14.85% | 37.78% | 4 | Conclusions This study developed a method for direct in-trap high-energy collision dissociation (HCD) within a dual-pressure linear ion trap mass spectrometer. By optimizing parameters using reserpine standard samples, the optimal conditions were established as the mixed collision gas of helium 1 sccm and nitrogen 0.1 sccm combined with the ion trap center section DC offset voltage configuration of -65 V, enabling efficient in-trap HCD. This approach also facilitated HCD-based MS³ analysis and successfully detected β-adrenergic receptor agonists requiring HCD fragmentation. The method eliminates the need for external collision cells, enhancing instrument compactness and reducing costs, while avoiding ion loss and time delays associated with ion transfer. Additionally, it enables MS³ analysis, a capability unavailable in triple quadrupole systems, and can be universally applied to any linear ion trap mass spectrometer, offering a simple and effective solution for HCD-based analyses. Acknowledgments This work is supported by the National Key R&D Program of China (No. 2022YFF0705001)，the NIM research foundation (No. AKYZZ2122) and the SAMR Scientific and Technological Programs (No. S2024MK0553).

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Authors Metrics & Citations Metrics Article Usage 355views 179downloads Citations Download citation Heyi Xu, You Jiang, Jie Xie, et al. Investigation of In-Trap High-energy Collision Dissociation Methodology and Dissociation Conditions Using Dual-Pressure Linear Ion Trap Mass Spectrometry. Authorea. 30 June 2025. DOI: https://doi.org/10.22541/au.175128515.56133563/v1 DOI: https://doi.org/10.22541/au.175128515.56133563/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.