Joint negative and positive mode development for combination LC-MS/MS Method
Combining mass spectrometry (MS) with chromatographic approaches is always desirable as MS is highly sensitive and specific compared to other detectors. MS 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 detectors to liquid chromatography separations was an obvious extension. However, the incompatibility of MS detectors with a continuous liquid stream was a significant challenge for its use in routine 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 mass spectrometry sample introduction in the 1980s. The electrospray ion source (ESI) was rapidly employed in LC/MS systems to analyze small molecules, proteins and peptides. Today, these systems have evolved into the LC-MS/MS method, with scientists using two MS quadrupole mass to charge filter units in tandem for more selective and specific analysis. Assessments through ESI involve incorporate detection in bothpositive and negative modes. Scientists may combine these modes and increase the dynamic range by rapidly switching between positive and negative ion polarity modes to incorporate compounds with broader chemical diversity. Let us dive deep into the working and development of positive, negative, and polarity switching modes in mass spectrometry.
Negative And Positive Ion Mode Mass Spectrometry
Mass spectrometers convert the molecules into a charged ionized state. These ionized molecules and fragments are then 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. Electrospray ionization (ESI) is one of the most widely employed ionization systems in LC-MS/MS analysis. ESI coupled with liquid chromatography is capable of analyzing many crucial classes of biological analytes. ESI is suitable for moderately polar molecules, and hence they are a perfect match for analyzing drugs, metabolites, peptides, biomarkers, and xenobiotics.
The ESI LC-MS/MS method produces multiply charged ions with analytes intact under appropriate instrumental conditions. As outlined before, the LC-MS/MS method has two polarity modes, ESI MS negative mode, and positive ion mode. The difference being that negative ion mode charges analyte through deprotonation, while positive ion mode charges through protonation.
Small molecules with a single functional group generally give singly charged ions. So in positive ion mode, these singly charged ions can be the addition of a proton, and in the 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 Positive And Negative Ion Mass Spectrometry
Samples are introduced into the LC-MS system through an electrospray probe consisting of a metallic capillary. As sample flow is introduced, current is applied between the tip of the probe and the sampling orifice. In electrospray ionization, a high voltage is introduced to the capillary while holding the sampling orifice at low voltage. Application of heat and voltage to the probe create a fine consistent spray. At low LC flow rates, the potential difference is enough for creating the spray. However, additional nitrogen gas flow is necessary for higher LC flow rates. As the name suggests, ESI mode requires the compound to be ionized before analysis. This is accomplished with amenable mobile phase conditions, energy applied to the mass spectrometer capillary and source, or post-column solvent addition if necessary to aid in 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 density of surface charge 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 analysis. Hence, ESI is the preferred choice for analyzing proteins, peptides, and biopolymers.
Developmental Conditions For ESI Mass Spectrometry
Low flow rates are ideal for achieving the best sensitivity in 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 ionization of the analytes of interest. Acidic pH is suitable for basic compounds analyzed in positive ion mode, while basic pH is ideal for acidic analytes analyzed in negative ion mode. However, some exceptions can be made depending on the method objectives and compound chemistry.
Volatile buffers are generally preferred in ESI LC-MS/MS 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 supress 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 also prevent the ionization of test analytes present in smaller concentrations. Moreover, care should be taken with LC 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 matrix effects in ESI LC-MS/MS analysis. 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 Positive And Negative Mode Mass 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-MS/MS analysis. Some potential alternatives for gas supply include liquid nitrogen dewars, nitrogen generators, 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 instrument. MS contamination due to the soldering flux and inadequate maintenance of N2 filtration/purification devices will result in an unusable detector.
Long-term 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 quality of LC columns. Contaminants such as solvent and oil vapors are not visible and are genuine concerns during LC-MS/MS analysis. Other sources of contamination include samples and solvents from pre-runs, buffers, and surfactants.
Adequate maintenance of both the HPLC unit and MS detector is critical for successful analysis. Routine maintenance generally consists of cleaning the source. This cleaning includes the source enclosure, the probe, and the sampling orifice. A dirty source affects the quality of the signal and risks further damage to the detector. This degradation is due to contaminated ions and reduction of ion transmission and may affect the voltages and transmission. A curtain gas protects the sampling orifice. However, after running the system with a phosphate bufferor 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 frequency of cleaning depends on the nature of samples, system usage, and source design. However, technicians should vent the system periodically. If system contamination occurs, further maintenance may be required resulting in the need for system venting, cleaning, re-establishment of vacuum, and performance verification, resulting in significant down time.
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 LC-MS/MS instruments.
Selection of mobile phase and eluent for MS has certain constraints. The requirements for MS eluents are different from other detectors. Primarily, the eluent must be appropriate for ionization and solute volatility is preferred. The choice of eluent 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 instrument, and hence, volatile organic acids are ideal for 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.