A Concise Review on Hyphenated Techniques: Liquid Chromatography Coupled Mass Spectroscopy (Lc-Ms/Ms)

Pairing or combining two distinct analytical approaches with the aid of an appropriate interface is known as a hyphenated technique. Spectroscopic techniques are mostly coupled with chromatographic techniques. Chromatography was used to separate the pure or almost pure fractions of chemical components in a mixture, while spectroscopy provides specific information that can be used to identify the components using standards or library spectra. The hyphenated technique is the result of integrating an internet-based spectroscopic detection technology with the separation approach. ^[1] Mass spectrometry/liquid chromatography, or LC/MS, is quickly taking the lead as the instrument preferred for liquid chromatographers. It is an excellent analytical method that combines mass spectrometry's detection specificity with liquid chromatography's resolving capability. ^[2] In laboratory medicine, it is still clear that LC-MS/MS is developing from the from the creative stage we discussed a decade ago to typical setups like automated analyser solutions that are frequently observed in contemporary laboratory conditions, it is still clear that LC-MS/MS in medical research is evolving. ^[3]

In conclusion, fundamental research findings that were used to the development of a method, piece of machinery, or measurement platform during a creative phase of technological development can occasionally be "forgotten" when they become commonplace. In this sense, immunoassays have changed from the research settings where they were initially developed and used. ^[4] In general, LC-MS is not widely used; less than 1% of studies conducted worldwide for MS-based laboratory diagnosis probably employ it. The relative numbers are significantly higher in specific application sectors, such toxicology and therapeutic drug monitoring (TDM), because there are fewer alternatives accessible. About 70% of organization taking part in proficiency testing employs LC–MS/MS for result creation when evaluating the immunosuppressive TDM. ^[5] It is an analytical method which fuses the physical separation abilities of liquid chromatography (often called HPLC) with the mass measuring powers of mass spectrometry. It is an analytical approach that combines mass spectrometry's mass measurement abilities with liquid chromatography's (also known as HPLC) physical separation capabilities. LC-MS is a strong method employed in many applications because of its exceptional sensitivity and selectivity. It is frequently used in pharmacokinetic studies of medications and is the most popular technique in the field of bioanalysis. ^[6] Numerous LC-MS systems with a variety of interface choices are currently available on the market. Enough liquid nebulization and vaporization, sample ionization, solvent vapor removal, and ion extraction into the mass analyser are provided by the interface design. In the context of natural product analysis, atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) are the two most widely used interfaces. The latter is known as "the chromatographer's LC-MS interface" because of its high solvent flow rate capabilities, sensitivity, linear response, and versatility. These interfaces can be used with a variety of analyser types, such as quadrupole, ion trap, and TOF. Each of these analysers offers a different level of mass accuracy and resolution, though. The LC-UV-MS mode also allows for the utilization of continuous-flow FAB (LC-CF-FAB) and thermospray (LC-TSP-MS) interfaces. Because it allows an aqueous phase to enter the mass system at flow rate of 1-2 ml/min, which is commensurate with the flow rates commonly employed in phytochemical analysis, the TSP interface has been shown as being the ideal one for phytochemical analysis. ^[7]

Because the recently developed API-based methods cause moderate ionization, they can be used in conjunction with LC-tandem MS, which is enabled by a triple quadrupole system, or fragmentation-induced collisions in the interface itself for structure elucidation studies. Through the use of tandem mass spectrometry and stable isotope internal standards, it can be applied to biological molecules and, by optimizing the technique to lessen the effects of ion repression, very sensitive and accurate procedures can be expanded. Between the drug development and discovery stages, technique validation is essential. Mass spectrometry and the physical separation of liquid chromatography, commonly referred to as HPLC, are combined in an analytical technique called LC-MS. A classic automated LC-MS system includes of an auto sampler, mass spectrometer, LC system, and double three-way diverter. The diverter usually acts as an automatic switching valve to send unwanted parts of the eluting from the LC system to trash before the sample enters the MS. ^[8]

PRINCIPLE

Principles of Liquid Chromatography-Mass Spectrometry (LC-MS):

Analyte separation using interactions with the stationary phase and mobile phase is the foundation of liquid chromatography (LC). Different retention times are the result of analytes partitioning between phases.

Ionization of analytes into charged particles (ions) is the fundamental idea behind mass spectrometry (MS). Ions are separated according to the mass-to-charge ratio (m/z).

Interface between LC and MS:

1. Ionization via Electrospray (ESI)

2. Chemical Ionization at Atmospheric Pressure (APCI)

3. Photoionization of Atmospheric Pressure (APPI)

Workflow for LC-MS:

Fig1: Workflow For LC-MS

Coupling of Liquid Chromatography with Mass Spectrometry

When a liquid was introduced into a mass spectrometer's high vacuum at a flow rate of 1 milliliter per minute and then evaporated, the high vacuum would instantly break down, making the coupling of HPLC with mass spectrometry a highly delicate and complicated process for a long time. In order to get over this innate incompatibility, numerous interfaces were created, each with unique benefits and drawbacks. ^[13]

We have not been able to obtain molecular ions for large nonvolatile molecules that are neutral in solution; however, ion-molecule reactions with reagent ions, either generated by gas-phase processes started by electron ionization or by thermospray from the buffer, can ionize molecules that are slightly volatile and thermally stable. In this operating mode, the system behaves remarkably similarly to the results of "desorption chemical ionization. ^[14] With TSP, a heated capillary was used to deliver the eluent into the vacuum, and a cold trap was used to extract the solvent vapor that was produced very challenging. For instance, the analyte was ionized by protonation using volatile additions such ammonium acetate. The first interface designed to deal with thermolabile substances was TSP. The continuous-flow fast atom bombardment (CFFAB) interface was an additional interface.^[15]

A small amount, roughly 5 ml/min, was divided into the mass spectrometer after a matrix with low volatility, like glycerol, was introduced to the HPLC eluent (2–5%). The dissolved analytes were left behind in a thin layer of glycerol after the solvent had evaporated from, say, a metal frit. The analytes were desorbed from this film by being bombarded with caesium ions or energetic xenon atoms. Although CFFAB was a rather sensitive technique, it was challenging to keep the glycerol film renewing steadily and maintaining a nice peak shape. Because the particle beam (PB) interface provides typical EI spectra that can be found in commercially available libraries, it is still rather significant. ^[16] About ten years ago, the so-called atmospheric pressure ionization (API) interfaces were introduced, and HPLC/MS became a success story. The HPLC eluent is evaporated outside the mass spectrometer via API interfaces. In order to eliminate the need to pump off massive amounts of gas, the analyte is also ionized outside the spectrometer at atmospheric pressure. Only the ions produced are then put into the mass spectrometer. Consequently, it is now simple to integrate mass spectrometric detectors with liquid chromatographic separations. Electrospray ionization (ESI) is the most popular interface technique, and APCI comes in second. ^{[17, 18]}

INSRUMENTATION

Fig 2: Instrumentation of LC-MS

Crucial Components of an LC-MS System:

1. Mobile phase reservoirs: How Mobile Phase Reservoirs Are Used in High Performance Liquid Chromatography. The pump will push the mobile phase solution into the column after it has been transported by the mobile phase reservoir. This movement is caused by the gravitational pull. When employed as mobile phase in LC, the solvents and solutions are stored in solvent reservoirs. These reservoirs are basically feed-receiving glass bottles or flasks
2. Pump: Provides a steady flow rate of the solvent mixture, or mobile phase.

Three typical pump types

1. Pumps with constant pressure: Because of a constant pressure, they, as their name implies, produce a constant flow rate in column. The low pressure of a gas inside a cylinder is used to generate the high pressure of the mobile phase that is required.
2. Syringe type pumps:
3. Reciprocating piston pumps:

3. Mixer
4. Degasser: Four methods used for degassing in HPLC are: 

1. Sonication
2. Vacuum
3. Helium sparging
4. In-line degassing.

5. Autosampler: Injects samples into the LC system automatically.
6. Column: Divides the sample's constituent parts according to how they interact with stationary phase. Normal phase, reverse phase, ion exchange, and size exclusion columns are the four primary varieties of HPLC columns. Every form of column is appropriate for a variety of applications and has pros and cons of its own.

The primary use of size exclusion HPLC columns is in the separation of proteins and carbohydrates. Racemic mixtures can be resolved using a chiral HPLC column; additional HPLC column types include displacement, affinity, and ion exclusion chromatography columns.

7. Detector: Due to its high sensitivity, ease of use, and superior durability, the ultraviolet (UV) or visible (VIS) detector is the most often used in HPLC (1). There are three major types of UV/VIS detectors are diode array detectors, fixed wavelength detectors, and variable wavelength detectors

Other

1. Fluorescence
2. Refractive index
3. Conductivity
4. Flame ionization detector

5. Interface

Ionization Source: Converts the analytes from the liquid phase to gas-phase ions. Common ionization techniques include:

1. Electrospray ionization (ESI): Soft ionization technique suitable for polar and ionic compounds.
2. Atmospheric pressure chemical ionization (APCI): Soft ionization technique suitable for less polar compounds.
3. Matrix-assisted laser desorption/ionization (MALDI): Hard ionization technique suitable for large biomolecules.

Mass instrumentation

1. Vaporization chamber: Simply said, a mass spectrometer is made up of an input chamber that is used to inject and evaporate the substance to be studied. All of its parts function under a high vacuum. After entering an ionization chamber, the gaseous molecules are subjected to a high-energy electron beam. Among other things, the electron beam produces a molecular ion, which is a positively charged molecule, created when one electron is removed from the molecule. After then, the molecule ion may fragment into smaller pieces. The pieces that are positively charged
2. Chamber of ionization: In mass spectrometry, a variety of ionization techniques are employed. Fast Atom Bombardment (FAB) and electron impact (EI) are the traditional techniques that the majority of chemists are familiar with.

Ionization Technique

Fig 3: Ionization techniques

3. Ion acceleration chamber: Any ion with the exact same kinetic energy is driven with an electric field into the "field-free" drift zone, which is devoid of electrical fields. An electric field is used to accelerate ions farther from the ion source.
4. Mass Analyzer: uses the m/z ratio of the ions to separate them. Typical mass analyzers consist of:

1. Quadruple: a straightforward and reliable tool for monitoring specific ions.
2. Triple Quadrupole: Used for quantitative analysis, this tool is extremely sensitive and selective.
3. Time-of-Flight (TOF): Used for precise mass measurements, it has a high mass accuracy and resolution.
4. Ion Trap: Used for qualitative as well as quantitative investigation, this tool has high sensitivity and adaptability.
5. Fourier Transform Ion Cyclotron Resonance (FT-ICR): Ultra-high mass accuracy and resolution, used for complex mixture analysis.

5. Detector: Detects the separated ions and generates a mass spectrum.

Some of the commonly used types include 

1. Electron multipliers (EM)
2. Faraday cups (FC)
3. Photomultiplier conversion dynodes
4. Array detectors.

OPTIMIZATION OF LC-MS METHOD

Resolution and peak shape, plate counts asymmetry, capacity factor, elution time, detection limits, limit of quantification, and overall ability to quantify the particular analyte of interest are among the initial sets of conditions that have developed from the first stages of development and are enhanced or maximized during the optimization stage. When developing a method, the following parameters need to be optimized:

Fig 4: Optimization parameters

1. Selection of mode of separation: The main determinant of the mode of separation is the analyte's nature. Reverse phase is the most favored mode for the separation of polar or moderately polar substances. The mobile phase seems significantly polar in nature than the stationary phase while in reverse phase mode. Electrospray ionization (ESI) was used for detection; a triple quadrapole mass spectrometer with an ESI interface allowed for both positive and negative ionization modes of operation. MS/MS detection is provided by the selective reaction monitoring mode (SRM/MRM). Techniques for gathering and displaying LC/MS data the mass spectrometer are usually configured to scan a particular mass range. This mass scan could be relatively narrow, as in selective ion monitoring, or it can be extensive, as in complete scan analysis. The following are the most common ways to obtain LC/MS data:

1. The standard total ion current plot (TIC) is produced by full scanning acquisition.
2. SIM, or selected ion monitoring.
3. Monitoring numerous reactions (MRM) or selected reactions (SRM).

2. Selection of column: The column is the central component of the chromatographic apparatus. High separation capability of column material (micro particles, 5-10 μm in size) packed to allow for the maximum number of theoretical plates. The most common ingredient used in the production of packing materials is silica (SiO2, H2O). It is composed of an upright, three-dimensional network of siloxane links (Si-O-Si) with interconnecting pores. As a result, a large variety of commercial items are accessible, with particle sizes ranging from 3 to 50 μm and surface areas between 100 and 800 m2/g. Silica's polarity is attributed to its sialon groups, which are used in adsorption chromatography with non-polar organic eluents. Although materials with C4 and C8 chains are also available, octadecyl-silica (ODS-Silica), which has C18 chains, the most widely used.
3. Mobile phase selection: Selectivity in LC is typically changed by altering the stationary phase's functionality or the composition of the mobile phase. Optimizing the composition of the mobile phase by varying the solvent type and strength, buffer type and concentration, and pH has proven to be the most effective and practical solution in the short term, especially for the study of ion compounds. The most often used solvents in LC/ESI-MS are acetonitrile admixtures, water, isopropanol, and methanol. ^[20]

Optimization of Mass Spectrometric Parameters

• Ionization Source: Ion formation and detection efficiency are affected by the ionization source selection (e.g., electrospray ionization, atmospheric pressure chemical ionization). It ought to be customized to the APS's physicochemical characteristics.
• Enhancing the specificity and sensitivity of the approach is possible by optimizing collision energy and mass transitions. To select the target analyte ions and increase signal-to-noise ratios, collision-induced dissociation (CID) and selective reaction monitoring (SRM) are frequently employed. ^[21]

Introduction to Bioanalytical Method Developmen

Medication concentrations in biological samples are precisely measured thanks to bioanalysis, which is essential to medication development. Due to its advantages over conventional techniques, including increased sensitivity and specificity, LC-MS/MS has become more and more popular in recent years. The assessment and interpretation of bioavailability, bioequivalence, and pharmacokinetic data are made easier by the use of bioanalytical techniques for the quantitative determination of medications and their metabolites in biological matrix.^[22]

Fig 5: Biological matrix

The following procedures are used to construct the LC-MS method: ^[23]

Fig 6: Construction the LC-MS method

Fig 7: Validation parameters

1. Accuracy: 

Method Comparison: Examine the outcomes of the bioanalytical approach in comparison to a reference or accepted methodology. To evaluate agreement, use statistical techniques such as Bland-Altman plots or regression analysis.

Studies that are spiking:  Compare the measured concentrations with the anticipated concentrations after adding known quantities of the analyte to the matrix (spiked samples). Analyze matrix effects and recovery during the spiking studies.

2. Precision:

Intra-day Precision: Examine several duplicates of the same sample in the same day under identical circumstances. Determine the results' standard deviation (SD) or relative standard deviation (RSD). 

Analyze duplicates of the exact same sample on various days or by various analyzers to determine intermediate precision, also known as inter-day precision. Add variances like various analysts, instruments, and days. Compute the results' SD or RSD.

3. Sensitivity

In bioanalysis, sensitivity is the analytical method's capacity to precisely identify and measure analyte concentrations at low levels. Sensitivity is essential for identifying traces of active pharmaceutical substances (APS) in biological matrices in the context of bioanalysis, especially when working with pharmaceuticals.

4. Selectivity

The ability of an analytical technique to precisely quantify the analyte being studied in its presence of additional components is known as selectivity in bioanalysis. Selectivity reduces false positives or negatives and guarantees that the method's reaction is unique to the target analyte.

5. Linearity

The potential of an analytical technique to give finding that are exactly proportionate to the concentration of analyte in the material being tested throughout a specific range is known as linearity. This implies that in bioanalysis, there should be a linear relationship among the concentration of analyte and the instrument response.

6. Range

The difference between the lowest and maximum analyte concentrations that have been shown to be reliably quantified with respectable precision and accuracy is known as the analytical method's range. It is a crucial parameter that establishes whether the procedure can be applied to particular concentration levels.

APPLICATIONS OF LC-MS/MS

Information like compound resolution, identity, and quantification can be obtained via LC-MS. Additionally; it facilitates chemical separation and purification. Other uses for LC-MS include.

Table no 1: Applications of LC-MS/MS.