LC-MS – Liquid Chromatography Mass Spectrometry
The LC-MS is a popular technique which is widely used in laboratories these days. This is so as GC-MS has long sample preparation time and extraction technique. They also need volatile analytes which need to be derivatized regularly. This volatility causes thermally weak analytes to be degraded before detection. That is why LC-MS is advantageous over the GC-MS technique. This is so because the LC-MS samples need lesser preparation time. Their instruments are also able to perform extraction on the instrument itself, with either plates or trapped columns. Extractions which are automated are successfully used to analyze methamphetamine and the drug’s metabolites in hair specimen along with MDA and MDMA from urine specimens with the help of LC-MS-Ms techniques. The benefit of LC-MS is that it doesn’t need to have expensive and time consuming procedures such as derivatization. Moreover, it can analyze polar and thermal compounds conveniently.
Another important reason for creating LC-MS is that the two techniques successfully interface each other which have led to huge enhancements in the field of forensic toxicology especially the application of instruments. The upcoming topics discuss important techniques of LC-MS fragmentation and ionization while being applied to forensic sciences.
Atmospheric Pressure Ionization
LC-MS is a technique that needs analyte transition from the liquid phase at atmospheric pressure to the vacuum inside the mass spectrometer. In order to aid this transition, ionization of the analytes takes place before they enter the mass spectrometer. Therefore, a special interface is made possible so that the transfer is facilitated easily.
There are two important functions of the LC-MS interface. Firstly, it removes the liquid medium and secondly, it transfers the analytes inside the mass spectrometer. Heater and gas nebulizers tend to dissolve the liquid phase especially by placing the nebulizer in optimum position with reference to the capillary. The instrument manufacturers don’t align the spray in front of the capillary. This is so because the capillary is a glass rod having metal coated on either ends and positions itself as a passageway between the interface region and the vacuum region inside the mass spectrometer. So, a potential difference is created between the capillary ends which cause the transfer of ionized analytes from the interface into the mass spectrometer. The latest interfaces made these days are normally perpendicular or orthogonal to the capillary, thereby causing a reduction in the amount of solvent and other material to enter the mass spectrometer. This tends to reduce the background interference and enhances the sensitivity of the process.
The second important function of the interface is the ionization of the analytes. This ionization can be done is numerous ways which depend on the type of interface present. The ionization can be done by hanging the sample’s pH utilizing acid-base chemistry. Many alkaline drugs which contain primary or secondary amines would take hydrogen ion under acidic environment and impart a positive charge on themselves. For instance, methamphetamine is a compound having a pKa of 9.9 in acidic phase like 0.1% acetonitrile/formic acid and a secondary amine group. The ionization of this drug takes place in the formic acid solution where it forms a pseudo-molecular ion [M+H]+. Another method via which ionization takes place is when charge transfers from gas molecule to the analytes via chemical ionization. Yet another method for ionization is the technique called photo-ionization in which UV lamp emits high energy containing photons which ionize vaporized analytes. A latest development in this field is the use of a laser that can vaporize and ionize the analytes present. All of the techniques mentioned here are utilized in LC-MS and LS-MS-MS techniques. However, their usage depends on the molecular weight and the polarity of the molecule that is being used.
Electro-Spray Ionization (ESI)
ESI – Electro-spray ionization is one of the common techniques that are a part of the atmospheric pressure ionization developed to be used in forensic sciences. The latest developed electro-spray interface tends to have pneumatically aided nebulization which increases the flow rate and solvent volume. There are 3 important parts of the electro-spray interface: nebulizer, desolvation assembly and repeller/mesh electrodes. The nebulization is done via nitrogen which enters the ESI to create an aerosol of charged droplets. The desolvation assembly causes the heated gas to evaporate and shrink the droplet so that the surface tension and the charges of the droplets cannot be further decreased in size. This point is known as ‘Rayleigh limit’ and the explosion of the droplets is referred as Coulombic explosion. This procedure continues to take place till all the solvents are evaporated and the free ions remain in the mixture. This is depicted in Figure 3.3. The ions then go into the capillary via the electrostatic potential difference that is created between the capillary ends and the mesh electrode which is placed at the orifice on the opposite side of the spray. The ESI source and its parts are depicted in Figure 3.4.
The electrostatic spray doesn’t produce any ions, the ions are already present in the solution. The electro-spray is a procedure in which ions are taken out from the solution and get transported into the mass spectrometer via the electrostatic potential. Varying according to the pKa of the compounds and the pH of the mobile phase, the ions can either have a positive or a negative charge.
Figure 3.3 and Figure 3.4 are depicted below.
Many latest ESI phases can analyze the positive and negative ions together or individually. The ESI is therefore, used for polar to mildly non polar drugs that can are charged in solution having acid-base pH chemistry. The SI has a charged mass range of around 3000 Dalton (Da). The alkaline based compounds are perfect for electro-spraying as they are easily charged and many drug plants contain alkaloids due to which electro-spray is invaluable for forensic science. The majority of ESI applications are used to analyze large molecules like enzymes and proteins. These molecules weigh around 10-200 kDa which multiple charge in the solution to produce ions with m/z having a range of mass spectrometer. The deconvolution software finds the structural identity and the molecular weight of the compound as well as the cluster of peaks which are produced by many charges.
Atmospheric Pressure Chemical Ionization
Another ionization technique which is complementary to ESI is known as APCI – Atmospheric Pressure Chemical Ionization. This technique is used to analyze low to medium polar molecules which are vaporized easily. In ESI, the compounds have multiple charges where as in APCI, the compounds have single charged ion. That is why, the mass range for APCI is constrained by the range of the mass spectrometer, being less than 3000 Da.
The introduction of APCI sample is quite similar to ESI. The APCI interface has four components i.e. nebulizer, corona needle, vaporizer and the desolvation module. Figure 3.5 depicts the APCI interface. The liquid medium enters nebulizer and passes through the needle assemblage. High pressure nitrogen (~60 psi) is blown via the nebulizer into the needle assembly which blasts the mobile liquid medium in a fine aerosol. The nebulized gas then carries the aerosol medium and analytes in a hot vaporizing tube having a temperature between 200-400°C. The temperature is optimized to have maximum solvent vaporization and decreases the thermal decomposition that occurs. The vaporized medium and its analytes are ionized with the help of corona needle discharge which is present at the end of the vaporization tube. A field of electrons is created by the corona needle that protonates the gas medium solvent while is leaves the tube. The charge is transported into the analytes by a process which is similar to methane-positive ionization in GC-MS. Varying according to the analyte and the application, the corona needle produces negative or positive ions. The analyte dependant variables include vaporization temperature, corona current and the nebulizer pressure which are optimized by FIA – Flow Injection Analysis.
The APCI is used for analyte having intermediate polarity and weight which doesn’t have acidic or basic sites. The samples which have compounds like esters, ketones, alcohols, aldehydes and hydrocarbons are analyzed using APCI. This is also correct for compounds which show sensitivity to acid/base chemistry and show bad response to electro-spray. The APCI can sustain high flow rate and can lodge a broader variety of solvents in contrast to ESI.
Some provisions should be considered before using APCI effectively. The compounds should be volatile enough to be vaporized and ionized. The molecular weight of the compound should be below 3000 Da as APCI produces only single charged ion. These two limitations prevent large, polar molecules like proteins and peptides. The high temperature of the vaporization tube degrades thermally labile molecules such as steroids, etc. Combined with ESI, many drugs and compounds which are tested in forensic laboratories like many legal and illegal drugs can be analyzed with APCI.
Atmospheric Pressure Photo-Ionization (APPI)
APPI – also known as Atmospheric Pressure Ionization uses a photon-releasing light source which ionizes the analytes. This is shown in Figure 3.6. The APPI has a gas discharge lamp which releases UV photons at prominent energy levels which are particular to the gas type used. The gases commonly used for APPI are: Krypton (10.0 & 10.6 eV), Xenon (8.4 eV) and Argon (11.2 eV). The analytes will ionize if their energies of ionization are lesser than those which are released by the source lamp. Also, the non polar analytes appear in the form of radical molecular ions (M+*) where as the polar compounds appear as pseudo-molecular ions [M+1]+. Depending on the set up of the mobile medium and analyte’s polarity, [M+*] which is created can accept hydrogen from the MP – mobile phase which produces [M+1]+ ions.
M + hv -> M+* + e–
M+* + MPH -> [M + 1]+ + MP*
APPI operates as both positive and negative ions. In the positive ion mode, the phase contains solvents like methanol which can donate hydrogen molecules easily. A popular choice for the mobile phase is water/acetonitrile. But, since there is no hydrogen for free donation, therefore this combination is not appropriate for APPI. The water acts like a strong base in the gas medium which has a strong attraction to the proton rather than solvent gas else the ionization efficiency is extremely reduced. In a negative mode, the gas of the reagent should have a strong attraction for protons or are able to capture electrons.
The APPI is not restrained by the acid/base chemistry or the volatility of the compounds. Rather, it is used to completely analyze the non polar to moderate polar compounds which are not open to either APCI or ESI. Low energy is used for ionization which can generate ions which are doubly charged, therefore increasing the range of the mass spectrometer, which is better than APCI but not as high as compared to ESI.
The compounds which are ionized directly or dopants are used to transfer charges indirectly. The direct ionization takes place if the ionized compound has ionization energy which is lesser than the photon. The dopants are the chemicals that are added to raise the ionization efficiency of APPI for compounds which don’t ionize easily or lose their ionizing capacity. The dopants are added to the nebulizer in the mobile medium and possess low ionizing energy. That is why they are quickly photonized and are able to transfer their charges to the compounds of their interest.
D + hv –> M+* + e–
D+* + M –> [M + 1]+ + D
D+* + M –> M+* + D
Acetone, Toulene and Anisole have been used quite successfully as dopants. Many APPI methods observe their sensitivity to be increased when dopants are used with an increase in their ionization efficiency. However, dopants can increase the formation of adducts, which complicates the mass spectrum’s interpretation.
Collision Induced Dissociation
CID – Collision Induced Dissociation is a technique used in IT-MS, LC-MS or MS-MS applications for the purpose of fragmentation. That is why CID is sometimes known as CAD – Collisionally Activated Dissociation. CID happens when the compounds that are being ionized gather speed in a limited area due to an electrical charge and then, collide with neutral gas molecules such as Helium, Argon or Nitrogen which causes them to fragmentize. FIA experimentation is done to optimize the parameters that are used in fragmentation. During the study of FIA, many parameters such as fragmentation voltage, nebulizing pressure, temperature and flow of the drying gas are changed with the sequential injection of standard solution. FIA determines the basic parameter for an individual drug compound and is normally performed before the analysis takes place. Ion abundance and its intensity establish the most favorable settings that are to be done. API ionization methods create even numbered electron ions. So in contrast to EI fragmentation, many losses of CID would either be even or neutral ions. The CID is dependent on analytes and the fragmentation that occurs depends on experimental parameters. Therefore, CID fragmentation is less energetic when compared to EI and at times, no fragmentation takes place. The full scan mass spectrum of the compound methadone s depicted in Figure 3.7 which utilizes three ionizing methods: PCI, ESI/Cid and EI. Extensive fragmentation is produced by EI where as PCI and ESI/CID do not produce any ionization.
CID can either occur in-source or in the mass analyzer. In the LC-MS method, the in-source CID is referred to as the fragmentation occurring before mass detection. When a potential difference is applied between the skimmer and the capillary end cap, the molecules speed up over a short distance which makes them collide with drying gas. The collisions cause fragmentation of the compounds. When the potential difference i.e. voltage of the fragmentor increases, the collision rate of the ions also increase, thereby causing varying levels of fragmentations. The fragmentor voltages are optimized to yield a particular fragmentation necessary for every analyte of interest. Different distances are found between skimmer and end cap, which vary according the different manufacturers. These differences affect the fragmentation of relevant molecules at certain voltages. The voltage of the fragmentor are not standardized as for EI which is fixed at 70eV, so even the same voltage is different instruments causes varying levels of fragmentation which depend on the manufacturer. Moreover, the molecular weight of the gas particles also affects the rate of fragmentation. Heavy gases like Argon have greater momentum, having a greater impact on the molecules to cause more fragmentation. The biggest limitation of LC-MS is the limited fragmentation caused due to in-source CID. Many computerized libraries have slowly developed and a lot of debate has occurred between the acceptable ions and ion ratios which are yet to be clearly defined. The L-MS data is more acceptable in European drug testing laboratories as compared to US arena as there as different legal systems working together.
In the technique of tandem quadruple MS, the second mass analyzer is used for CID to take place via increasing the pressure and speeding the ions which collide with gas molecules. Moreover, CID takes place in an ion trap in which energy is applied, causing the ions to get excited, which then collide with Helium gas to cause fragmentation. Ions are cooled and focused inside the trap walls. The mass analyzer CID passes on more specificity and efficiency to the collision as compared to the in-source CID. Before fragmenting, the ions are separated, which eliminates the chances of co-elution of ion interferences that might occur during in-source CID. That is why, the mass analyzer is the selected method while studying fragmenting pattern and identifying unknown.
Two Dimension Gas Chromatography – Mass Spectrometry
2D-GC-MS i.e. two dimensional gas chromatography – mass spectrometry is a technique that uses two gas chromatography capillary columns having different polarities to remove background interferences from matrices and increase their sensitivity. Due to an increased interest to test alternative matrices like oral fluid and hair, where detection limits are low, the 2D-GC-MS offers simple and inexpensive means to enhance the sensitivity of the instruments which exist in laboratories. This is also important for laboratories which run on limited budgets.
Enhanced sensitivity is also achieved by transferring a selected part of the eluent which contains the analytes into a second capillary column which is connected to a mass spectrometer. This is sometimes known as a ‘heart cut’ which is concentrated with cryotrap prior to its introduction into the second capillary column, which enhances its resolution and perks up the peak shapes. The eluent remaining before and after this segment is send for FID where its monitoring and evaluation takes place. Vastly improving the s/n, this technique improves the sensitivity of the method. It has also been quite successfully used for analyzing the 11-nor-∆9-tetracannabinol-9-carboxyli acid in hair specimen and MDMA and other compound concentration in plasma. In oral fluids, techniques are used to test the sample size which is normally less than 1mL and thus requires more sensitive methods to be used rather than conventional GC-QMS technique. In Figure 3.8 shows the increased sensitivity that a 2D-GC-MS can provide.