Joint negative and positive mode development for combination LC-MS/MS Method

Positive and Negative Mode in Mass Spectroscopy: LC-MS/MS Method | NorthEast BioLab

Combining mass spectrometry (MS) with chromatographic approaches is always desirable as MS is highly sensitive and specific compared to other detectors. Mass spectrometry tuning, a critical aspect, ensures optimal performance by adjusting parameters such as ionization voltage and collision energy. MS MS mass spec was commercially coupled with gas chromatography (GC) early in the 1970s, providing cheap and reliable detecting systems for clinical and biochemical laboratories. Still today, several laboratories use GC-MS for detecting and quantifying complex compounds in biological samples. GC, however, requires compound volatility or derivatization to infer compound volatility. Hence, the expansion of coupling with MS MS mass spectrometry detectors to liquid chromatography separations was an obvious extension. However, the incompatibility of MS MS mass spectrometry detectors with a continuous liquid stream was a significant challenge for its use in routine MS MS analysis. Bioanalytical scientists developed several interfaces to overcome this challenge during the 1970s, but all efforts were unfruitful.

This situation changed when Fenn developed the electrospray ionization source for MS MS spectrometry sample introduction in the 1980s. The electrospray ion source (ESI) was rapidly employed in LC-MS Mass Spec systems, ESI Mass Spec, to analyze small molecules, proteins, and peptides. Today, these systems have evolved into the LC Mass Spec MS method, with scientists using two MS MS mass spectrometry quadrupole masses to charge filter units in tandem for more selective and specific analysis. Assessments through ESI mass spectrometry involve incorporating detection in both MS positive and negative modes. Mass spectrometry tuning is essential to optimize its performance for different analytes and applications. Scientists may combine these modes and increase the dynamic range by rapidly switching between MS positive and ESI negative ion polarity modes to incorporate compounds with broader chemical diversity. Let us dive deep into the working and development of MS positive, negative, and polarity switching modes.

Negative And Positive Ion Mode MS MS Spectrometry

MS MS mass spec spectrometers convert the molecules into a charged ionized state. These ionized molecules and fragments are detected based on their mass-to-charge ratios. Scientists have a plethora of ionization and ion analysis systems at their disposal. However, some system configurations are more suitable than others. Mass spectrometry tuning is essential for optimizing system configurations, ensuring the most suitable setup for specific analyses. Electrospray ionization (ESI) is one of LC-MS Mass Spec analysis’s most widely employed ionization systems. ESI Mass Spectrometry, coupled with liquid chromatography, can analyze many crucial classes of biological analytes. ESI Mass Spec is suitable for moderately polar molecules; hence, they are a perfect match for analyzing drugs, metabolites, peptides, biomarkers, and xenobiotics.

The ESI LC-MS Mass Spec method produces multiply charged ions with analytes intact under appropriate instrumental conditions. The MS MS spectrometry method has two polarity modes: ESI Mass Spec negative and positive ion modes, each requiring precise mass spectrometry tuning. The difference is that the ESI negative ion mode charges the analyte through deprotonation, while the MS positive ion mode charges through protonation.

Small molecules with a single functional group generally give singly charged ions. So, in MS positive ion mode, these singly charged ions can be the addition of a proton; in the ESI negative ion mode, it can be the loss of a proton. On the other hand, larger molecules such as peptides and proteins have several charged functional groups. Multiple functional groups form an envelope of ions around the molecule. These different charged states help determine the analytes with high molecular weights. However, most triple-quadrupole mass spectrometers can scan up to approximately 4000 m/z, varying with make, model, and calibration specifications.

Electrospray Ionization In MS Positive And Negative Ion MS MS Spectrometry

An electrospray probe with a metallic capillary introduces samples into the LC-MS Mass Spec system. Mass spectrometry tuning is crucial for optimizing the performance of the system. As sample flow is introduced, current is applied between the probe tip and the sampling orifice. In electrospray ionization, a high voltage is introduced to the capillary while holding the sampling orifice at a low voltage. Applying heat and voltage to the probe creates a fine, consistent spray. The potential difference is enough to create the spray at low LC flow rates. However, additional nitrogen gas flow is necessary for higher LC Mass Spec flow rates. As the name suggests, ESI Mass Spectrometry mode requires the compound to be ionized before MS MS analysis. If necessary, this is accomplished with amenable mobile phase conditions, energy applied to the mass spectrometer capillary and source, or post-column solvent addition to aid ionization and signal sensitivity.

The electrical field at the capillary tip will form droplets of the ionized compounds. These droplets will either be positively or negatively charged depending on the polarity of the applied voltage. As the solvent evaporates, the droplet size reduces while the surface charge density increases. These differences increase the repulsion forces between charges until the droplet explodes. This entire process is repeated until the ions evaporate from the droplet. Depending on the analyte of interest, scientists can obtain several charged ions for MS MS analysis. Hence, ESI Mass Spec is the preferred choice for analyzing proteins, peptides, and biopolymers.

Developmental Conditions For ESI LC-MS Mass Spec

Low flow rates are ideal for achieving the best sensitivity in MS MS spectrometry. Although 1ml/min or higher flow rates can be achieved, they may reduce the signal-to-noise ratio. For improved response, the pH of the mobile phase should facilitate the ionization of the analytes of interest. Acidic pH is suitable for basic compounds analyzed in MS positive ion mode, while basic pH is ideal for acidic analytes analyzed in ESI negative ion mode. However, some exceptions can be made depending on the MS method objectives and compound chemistry.

Volatile buffers are generally preferred in mass spectrometry tuning for ESI LC-MS Mass Spec analysis. Though nonvolatile buffers can be used, they require timely removal of salt deposits and much more extensive instrument maintenance. Moreover, buffer concentration needs to be as low as possible because a higher concentration will create competition between the electrolyte and analyte ions and suppress the analyte response. For example, a charged species in excess will cover the droplet surface area and block the access of other ions to the surface. Additionally, high-concentration components may prevent the ionization of test analytes in smaller concentrations. Moreover, care should be taken with LC Mass Spec ion pairing agents because their presence impacts the formation of the spray and evaporation of droplets and may result in suppressed sensitivity.

Moreover, scientists must evaluate and reduce ESI Mass Spectrometry analysis matrix effects. In the presence of high salt concentrations or excess of other ionized analytes, a competition effect called “ion suppression” may happen with ionization. Hence, scientists must develop appropriate chromatographic separation to reduce matrix effects.

Technical Considerations For Developing MS Positive And Negative Mode MS MS Spectrometry

Laboratories must ensure the availability of appropriate power supply, backup generators, battery backups for power outages, clean compressed air, and nitrogen supply while planning a site for LC Mass Spec analysis. Potential gas supply alternatives include liquid nitrogen dewars, nitrogen generators, and liquid nitrogen facility tanks. The gas and plumbing for its delivery must be free from organic impurities, as they may be detectable on the mass spectra and contaminate the MS MS mass spec instrument, potentially causing ion suppression in mass spectrometry. Regular mass spectrometry tuning ensures the sensitivity and reliability of the instrument for precise analysis. MS contamination due to the soldering flux and inadequate N2 filtration/purification device maintenance will result in an unusable detector.

Long-term MS MS analysis requires a clean environment. Moreover, an adequate exhaust is necessary to avoid the dispersion of oil vapors and solvent vapors into the laboratory from the pumps required to maintain the mass spectrometer vacuum. A clean environment includes the lab, sample preparation, quality of buffers and solvents, and LC Mass Spec columns. Contaminants such as solvent and oil vapors are not visible and are genuine concerns during LC-MS Mass Spec analysis. Other sources of contamination include samples and solvents from pre-runs, buffers, and surfactants.

Advanced technologies such as UPLC mass spectrometry can enhance analytical capabilities, offering higher resolution and faster analysis times. Integration of UPLC with mass spectrometry enables improved separation efficiency and sensitivity, facilitating the detection of trace analytes and enhancing overall analytical performance.

Maintaining the HPLC unit and MS MS spectrometry detector is critical for successful MS MS analysis, including when utilizing UPLC mass spectrometry. Routine maintenance generally consists of cleaning the source. This cleaning includes the source enclosure, the probe, and the sampling orifice to prevent ion suppression in mass spectrometry. A dirty source affects the quality of the signal and risks further damage to the detector. This degradation is due to contaminated ions and reduced ion transmission and may affect the voltages and transmission. A curtain gas protects the sampling orifice. However, after running the system with a phosphate buffer or other nonvolatile solvents, the salt gets deposited on the source and needs to be removed frequently following each analysis. Technicians can disassemble the source housing and clean it without venting the system. The cleaning frequency depends on the samples’ nature, system usage, and source design, with a focus on mitigating ion suppression in mass spectrometry. However, technicians should vent the system periodically. If system contamination occurs, further maintenance may be required, resulting in system venting, cleaning, re-establishment of vacuum, and performance verification, resulting in significant downtime.

The roughing pump oil in the vacuum system needs to be changed on average every 6 months. In the case of electron multiplier detection systems, technicians must replace the electron multiplier typically after about 2-3 years based on performance and maintenance testing. Maintenance contracts for preventive maintenance and repair are highly recommended for complex analytical systems and reduce the downtime of MS MS mass spec instruments.

The mobile phase selection and eluent for LC Mass Spec have certain constraints. The requirements for MS eluents are different from other detectors. The eluent must be appropriate for ionization, and solute volatility is preferred. Eluent choice depends on the analyte of interest and mode of ionization. Using non-volatile acids such as HCl and methane sulfonic acid might damage the LC-MS Mass Spec instrument, and hence, volatile organic acids are ideal for MS MS analysis. Ideally, the buffer concentration must be as low as possible to reduce ion suppression. Finally, the HPLC column must provide adequate separation, ideally without using ion-pairing reagents and high-concentration buffers.