Potential of the Linear Ion trap Mass Spectrometer

Dr Manoj Pillai, Director, Application Support, SCIEX, India and Raghunath MV, Market Development Manager, SCIEX, India elaborate on the potential of QTRAP system for Impurity Profiling/ Genotoxic Quantitation

Liquid chromatography combined with atmospheric pressure ionization (API) tandem mass spectrometry (LC/MS/MS) is particularly suitable for the analysis of pharma compounds. Triple quadrupole systems have been widely used for quantitation due to their specificity, selectivity and high duty cycle in MRM mode. In addition to the MRM scan, triple quadrupole instruments can also perform selective scan functions like constant neutral loss and precursor ion scan which are useful for establishing structural relationship.  At the same time the full scan sensitivity of the ion trap based mass spectrometers make it better suitable for qualitative analysis compared to the quadrupole based mass analyzers. Hybrid instruments like QTRAPTM provide the capability and specificity of triple quadrupole scan functions and sensitivity of a ion trap operational modes present unique opportunities for the use in both quantitative and qualitative analysis.

Hybrid Triple Quadrupole Linear Ion Trap (QTRAP®) Systems provide a novel workflow for the screening and identification of a multitude of targeted analytes by combining selective MRM detection with a highly sensitive MS/MS scan using Q3 as Linear Ion Trap. In Information Dependent Acquisition (IDA) experiments, the detection of an MRM signal above a specified threshold automatically triggers an Enhanced Product Ion (EPI) scan. These EPI spectra are as sensitive and selective as MRM signals and contain the complete molecular fingerprint because of precursor ion selection in Q1, product ion generation in the collision cell (Q2), and ion accumulation in the LIT (Q3).

The rich product ion spectra are the result of the generation of fragment ions in the collision cell. These spectra can be searched against existing mass spectra libraries. The information saved into a full scan MS/MS spectrum allows identification with a higher degree of confidence minimizing the risk of potential false positive and negative detection. In addition, the improved cycle time for all confirmatory MRM transitions can be used to increase the dwell time of all other MRM transitions to improve S/N, resulting in better reproducibility and accuracy. Alternatively, additional compounds can be screened in the MRM survey scan.

The additional capabilities of the QTRAP system based on the IDA workflow are given below:

Triple quadrupole scan functionality

• MRM & dynamic range for quantitation
• Neutral loss scanning for structural relationship between patent and impurities
• Precursor ion scanning for structural relationship between patent and impurities
• Normal quadrupole style MS/MS fragmentation pattern

QTRAP Scan Functinality

• Superior full scan sensitivity in EMS, MS/MS and MS/ MS/ MS scan
• High resolution scanning (For isotopic pattren determination)
• Enhanced multiply charged scanning (Peptide analysis)
• MRM3 quantitation
• MRM and dynamic range for qunatitation
• Neutral loss scanning for structural relationship between patent and impurities
• precursor ion scanning for structural relationship between patent and impurities
• IDA triggered on-the-fly combination of classic QqQ and QTRAP specific scan modes enabling unique & powerful new LCMSMS workflows

Role of Quadrupole Based Mass Analyzer in Impurity Profiling/ Genotoxic Quantitation

• Best for low level Quantitation (Use MRM selective scan)
• Neutral loss and Precursor Ion scan – Best to find the structurally similar compound in impurity Profiling
• Low Full Scan sensitivity
• No accurate isotopic pattern determination
• Product Ion Spectra (Unit resolution)
• Both quantitative and qualitative analysis cannot be possible on typical Triple Quad based system.
• No MS/MS/MS for interpretation of Impurities
• Resulting in no confirmation of any impurities or degradation products

Benefits of QTRAP Based Mass Analyzer in Impurity Prolfing/ Genotoxic Quantitation

• Best Full Scan Sensitivity (Trap Mode)
• Best product Ion Scan (Trap Mode)
• MS/MS/MS (Trap Mode)
• All the QqQ scan function will remain same, hence best for Quantitation (Quadrupole Mode)
• Tools like Information Dependent Analysis (IDA) enables user to improve their throughput by generating MS and MSMS data in a single LC run, thus high throughput.
• Can perform high sensitivity quantitative experiments in combination with high sensitivity qualitative scans in one experiment, which is ideally suited to investigations of both expected and unexpected impurities.

Traditional QqQ vs QTRAP (IDA-DBS) Based Impurity Profiling

Workflow based experiments in QTRAPTM for Impurity Profiling

• Information dependant workflow (IDA)

1. IDA helps in the on the fly acquisition of the Full scan spectra (EMS), the isotopic pattern of the masses (ER), the fragmentation pattern (EPI) and can also do the MS/MS/MS.  This will be an ideal tool for unknown sample analysis as this can collect the detailed data from a single LC MS run.
2. Further IDA workflows can use the combination of scan functions that is pertaining to the triple quad and the trap thus improving the power of both qualitative and quantitative analysis.  This is unique to the hybrid QTRAP instrument.
3. The combination of scan functions from the triple quad and trap makes the identification workflow very selective and specific too.
4. IDA workflows can be used for general unknown screening, impurity profiling and metabolite identification, and for any simultaneous quantitative and qualitative work.

Real time algorithm for Impurity profiling with QTRAP

• Dynamic background substraction (DBS)
• Collision energy Spread (CES)

Dynamic Background substration (DBS): This is noval automated MS/ MS techniques for impurities analysis. Real time dynamic background substraction (DBS) is useful triggering tool for MS/MS in IDA experiments, on peaks of interest not on the background ion. The concept of DBS is to subtract the previous scan from the current one, before applying any other IDA selection criteria. This can eliminate the need for a second injection in order to acquire MS/MS.  This will be use full tool for characterizing the impurities present at very low level in API samples.

Collision energy Spread (CES): This real time algorithm uses three different collision energy to fragments the ion of interest. This reduce the optimisation step for enhanced product ion spectra and MS/MS/MS. This algorithm avoids reinjection of samples to get the best and rich production spectra.