Hybrid Linear Ion Trap Mass Spectrometer

Mass spectrometry has developed rapidly over the past decade and is now used in almost every field. In particular, in the life sciences, mass spectrometry has become an essential analytical tool for protein and small molecule applications.

From a technical point of view, most mass spectrometers cannot obtain all the information required, so two or more detection methods must be used simultaneously to achieve maximum sensitivity and throughput. This is true for both quadrupole and ion trap analysis. For some applications, it is difficult to obtain high-quality spectra with triple quadrupole devices because the sensitivity of product ions is usually not very strong. This lack of sensitivity can be overcome by QqTOF technology, which can provide additional mass spectral information, but true precursor and neutral loss scanning experiments and reliable quantitative analysis are difficult to achieve. Although ion trap mass spectrometers (MS) can perform multiple sensitive mass spectrometry (MSn) experiments to clarify reaction mechanisms and simplify spectral interferences, the previous problems are also unavoidable for 3D ion trap technology. However, 3D ion traps are prone to overloading (spatial charging effects), which can lead to mass drift, resolution loss, and nonlinearity of spectra and calibration curves. Therefore, 3D ion traps are not suitable for large-volume analysis. All of this has changed since the development of the first generation of hybrid linear ion trap/triple quadrupole mass spectrometers. The Q TRAP LC/MS/MS system from Applied Biosystems and MDS SCIEX, developed on the basis of the API 2000 LC/MS/MS platform, has been launched on the market and immediately received widespread popularity in the field of protein and drug development mass spectrometry.

API 2000 Liquid Chromatography-Mass Spectrometer

This equipment has more technical advantages than the traditional three-dimensional ion trap mass spectrometer. Since the linear ion trap has a storage capacity of about 45 times higher than that of traditional equipment, it is not very sensitive to spatial charging effects. At the same time, due to the enhanced product ion (EPI) detection system, the lower molecular weight part will not be ignored, and the sensitivity achieved by traditional multiple reaction modulation (MRM) can now be achieved through EPI scanning. Since the high sensitivity can be achieved simultaneously on the same device, the following work becomes possible: a) combining experiments that originally had to be performed on two different mass spectrometry technology platforms; b) easily obtaining data that was not easy to obtain before.

4000 Q TRAP LC/MS/MS

The development of the 4000 Q TRAP LC/MS/MS system has taken this technology a step further, with the same quantity requirements as the original product, but with a sensitivity increase of 10 to 50 times depending on the application. This equipment can provide rapid, automated, qualitative and quantitative analysis, with applications in the pharmaceutical, food, environmental, clinical research, forensics, toxicology and metabolic industries. The system also has a wider mass range (mass-to-charge ratio up to 2800), which enables the system to perform automatic analysis after mobile correction (PTMs) and quantitative detection of protein authentication and peptide bonds. The 4000 Q TRAP system has four different ion sources to choose from: TurboV ion source; DuoSpray ion source, which is a combination of TurboIonSpray and APCI in the same ion source, which can achieve rapid switching (100ms) between the two ionization techniques; Photospray source for atmospheric pressure photoionization and Nanospray source for on-line and off-line peptide bond and protein analysis. Several additional software packages are available, such as Metabolite ID for automated identification and metabolic profiling, Bioanalysis software, and ProID software for protein applications.

Mass Spectrometry in Forensic Toxicology

The power of this instrument can be seen in its use in routine forensic toxicology examinations, where the main objectives are as follows:

■ To detect and determine the presence of conventional and non-conventional drugs in blood, urine, and saliva samples
■ To obtain important structural information about the drug components in the same chromatographic separation as further evidence
■ To analyze extracts that may be related to any drug detected and provide positive evidence

In the past, this type of analysis was usually performed on a triple quadrupole instrument in MRM mode, analyzing a large number of selected target compounds. In addition, in order to obtain structural information, a three-dimensional ion trap instrument was required because the detection sensitivity of this instrument also has the capabilities of MS3. However, with the 4000 Q TRAP system, all information can be obtained from the system in a single test. The test process is controlled by the Information Independent Acquisition (IDA) software package, which has been integrated into the Analyst 1.4 software. IDA software links scan profile information with process selection, taking advantage of special detections such as ions and neutral losses and expanding the scope of data acquisition. Figure 2 shows a multi-stage experiment handled by IDA software during the complete cycle of LC analysis. In all IDA ion selection criteria, special mass and retention time (SMART) filters can be applied to include and exclude ions of interest. When MS/MS confirmation is required, it provides users with maximum flexibility in cases with the same atomic weight or in fast special analysis. Enhanced bombardment separation can be added as a further confirmation scan, which can obtain more accurate mass spectral information. This information is particularly effective for post-detection applications (precursor and neutral loss) with 1-5 charge ion determination and isotope ratio measurement, because ordinary detection in this case is usually difficult to obtain useful information. Because the 4000 Q TRAP system has the ability to collect three-dimensional mass spectral data, there are two levels of accessories in the loop. With the LINAC collision cell, the system can also perform multiple MRM transitions, so that a large number of compounds can be detected simultaneously without losing sensitivity. In the example below (see Figures 2 and 3), nine common sedatives were detected in a urine sample. A 5-minute liquid chromatography (LC) gradient was run at 500 μl/min and all nine sedatives were displayed, with MRM transitions for each. When a signal was detected during a specific MRM transition, the software automatically acquired enhanced product ion MS/MS spectra, providing structural information for each sedative. By performing experiments on a single system and using automated software tools such as IDA to perform analysis in a single LC run, sample analysis time was successfully compressed, and different MS platforms did not require changes in LC run conditions.

Similarly, the successful combination of sensitive triple quadrupole and ion trap detection in a single analysis has certain advantages in the identification and ranking of post-mobility modifications (PTMs), especially in phosphorylation. For phosphorylation analysis, a negative ion fragment with a mass-to-charge ratio of 79 was detected, representing the detachment of a single phosphate molecule from the phosphorus-containing peptide bond. This precursor ion scan was used for comprehensive detection in the automated IDA. Ions present in the precursor scan are then automatically analyzed in the positive ion with an enhanced decomposition scan and then analyzed using a high sensitivity MS/MS scan to identify phosphorylation peptide bonds and the location of phosphorylation. Pro ID software is used to monitor protein formation to understand each peptide bond and determine the location of peptide bond adjustments.