BIOANALYTICAL TECHNIQUES – AN OVERVIEW

 

Instrumentation:
The GC-MS is composed of two major building blocks: the gas chromatograph and the mass spectrometer. The gas chromatograph utilizes a capillary column which depends on the column's dimensions (length, diameter, film thickness) as well as the phase properties. The difference in the chemical properties between different molecules in a mixture will separate the molecules as the sample travels the length of the column. The molecules are retained by the column and then elute (come off of) from the column at different times (called the retention time), and this allows the mass spectrometer downstream to capture, ionize, accelerate, deflect, and detect the ionized molecules separately. The mass spectrometer does this by breaking each molecule into ionized fragments and detect the fragment [8].

GC-MS schematic:
These two components, used together, allow a much finer degree of substance identification than used separately. It is not possible to make an accurate identification of a particular molecule by gas chromatography or mass spectrometry alone. The mass spectrometry process normally requires a very pure sample while gas chromatography using a traditional detector (e.g. Flame ionization detector) cannot differentiate between multiple molecules that happen to take the same amount of time to travel through the column (i.e. have the same retention time), which results in two or more molecules that co-elute9. Sometimes two different molecules can also have a similar pattern of ionized fragments in a mass spectrometer (mass spectrum). Combining the two processes reduces the possibility of error.

Purge and trap GC-MS
For the analysis of volatile compounds a purge and trap (P&T) concentrator system may be used to introduce samples. The target analytes are extracted and mixed with water and introduced into an air tight chamber. An inert gas such as Nitrogen (N2) is bubbled through the water; this is known as purging. The volatile compounds move into the headspace above the water and are drawn along a pressure gradient (caused by the introduction of the purge gas) out of the chamber. The volatile compounds are drawn along a heated line onto a 'trap'. The trap is a column of adsorbent material at ambient temperature that holds the compounds by returning them to the liquid phase. The trap is then heated and the sample compounds are introduced to the GC-MS column via a volatiles interface, which is a split inlet system. P&T GCMS is particularly suited to volatile organic compounds.

Types of mass spectrometer detectors
The most common type of mass spectrometer (MS) associated with a gas chromatograph (GC) is the quadrupole mass spectrometer. Another relatively common detector is the ion trap mass spectrometer. Additionally one may find a magnetic sector mass spectrometer, however these particular instruments are expensive and bulky and not typically found in high throughput service laboratories. Other detectors may be encountered such as time of flight (TOF), tandem quadrupoles [9].

Ionization
After the molecules travel the length of the column, pass through the transfer line and enter into the mass spectrometer they are ionized by various methods with typically only one method being used at any given time12. Once the sample is fragmented it will then be detected, usually by an electron multiplier diode, which essentially turns the ionized mass fragment into an electrical signal that is then detected. Electron ionization: In the electron ionization (EI) the molecules enter into the MS (the source is a quadrupole or the ion trap itself in an ion trap MS) where they are bombarded with free electrons emitted from a filament, not unlike the filament one would find in a standard light bulb. The electrons bombard the molecules, causing the molecule to fragment in a characteristic and reproducible way. This "hard ionization" technique results in the creation of more fragments of low mass to charge ratio (m/z). Hard ionization is considered by mass spectrometrists as the employ of molecular electron bombardment, whereas "soft ionization" is charge by molecular collision with an introduced gas. The molecular fragmentation pattern is dependent upon the electron energy applied to the system, typically 70 eV (electron Volts)[10].

Analysis
A mass spectrometer is typically utilized in one of two ways: full scan or selected ion monitoring (SIM). The typical GC-MS instrument is capable of performing both functions either individually or concomitantly, depending on the setup of the particular instrument. The primary goal of instrument analysis is to quantify an amount of substance. This is done by comparing the relative concentrations among the atomic masses in the generated spectrum. Two kinds of analysis are possible, comparative and original. Comparative analysis essentially compares the given spectrum to a spectrum library to see if its characteristics are present for some sample in the library. Another method of analysis measures the peaks in relation to one another. In this method, the tallest peak is assigned 100% of the value, and the other peaks being assigned proportionate values. All values above 3% are assigned[ 25]. The total mass of the unknown compound is normally indicated by the parent peak. The isotope pattern in the spectrum, which is unique for elements that have many isotopes, can also be used to identify the various elements present.

Full Scan MS
Full scan is useful in determining unknown compounds in a sample. It provides more information than SIM when it comes to confirming or resolving compounds in a sample. During instrument method development it may be common to first analyze test solutions in full scan mode to determine the retention time and the mass fragment fingerprint before moving to a SIM instrument method.[11]

Selected ion monitoring
In selected ion monitoring (SIM) certain ion fragments are entered into the instrument method and only those mass fragments are detected by the mass spectrometer. The advantages of SIM are that the detection limit is lower since the instrument is only looking at a small number of fragments (e.g. three fragments) during each scan. More scans can take place each second[12].

Gas chromatographs
Routine maintenance operations used include checks on the septum, injector liner, gas pressures and inlet filters (e.g. oxygen scrubber, moisture trap and charcoal trap), baseline signal level and background noise. Depending on the degree of usage of the instrument, it is sensible to have a routine maintenance program involving weekly change of the septum and injector liner.

Figure 2: GC/MS Instrument[24]

High Performance Liquid Chromatography (HPLC)
High performance liquid chromatography is a form of column chromatography used frequently in bio chemistry and analytical chromatographic packing material (stationary phase),a pump that moves the mobile phase(s) through the column, and a detector that shows the retention time of the molecules, retention time varies depending on the interactions between the stationary phase, the molecules being analyzed and the solvent(s) used. Bionalytical method development is the process of creating a procedure to enable a compound of interest to be identified and quantified in a matrix. By using biological products can be measured by several methods and the choice of bioanalytical method involves several considerations of quantitative or qualitative measurement, and precision are required with necessary equipment. The bioanalytical chain describes the process of method development by biological samples includes sampling, sample preparation, separation, detection and evaluation of the results.[13]

Some General procedures for sample preparation are
liquid/liquid extraction
solid-phase extraction (SPE) and
Protein precipitation.

Liquid – Liquid extraction
It is based on the principles of differential solubility and partitioning equilibrium of analyte molecules between aqueous (the original sample) and the organic phases. Now a day’s Liquid-Liquid extraction has been replaced with advanced and improved techniques like liquid phase micro extraction, single drop liquid phase micro extraction and supported membrane extraction. Liquid – Liquid extraction generally involves the extraction of a substance from one liquid phase to another liquid phase [14].

Solid Phase Extraction (SPE)
Solid phase extraction is selective method for sample preparation where the analyte is bound onto a solid support, interferences are washed off and the analyte is selectively eluted. Due to different sorbents, solid phase extraction is a very powerful technique. Further Solid phase consists of four steps they are; conditioning, sample loading, washing and elution.

Protein Precipitation
Protein precipitation is often used in routine analysis to remove proteins. Precipitation can be induced by the addition of an organic modifier, a salt or by changing the Ph which influence the solubility of the proteins [15]. The samples are centrifuged and the supernatant can be injected into the HPLC system or be evaporated to dryness and thereafter dissolved in a suitable solvent. A concentration of the sample is then achieved. However, the protein precipitation technique is often combined with SPE to produce clean extract. Salts are other alternative to acid organic solvent precipitation. This technique is called as salt induced precipitation. As the salt concentration of a solution is increased, proteins aggregate and precipitate from the solution [16].

Conditioning
The column is activated with an organic solvent that acts as a wetting agent on the packing material and solvates the functional groups of the sorbent. Water or aqueous buffer is added to activate the column for proper adsorption mechanisms.

Sample Loading and elution
Distribution of analyte–sorbent interactions by appropriate solvent, removing as little of the remaining interferences as possible. Typically, sorbents used in SPE consists of 40μm diameter silica gel with approximately 60 A0 pore diameters. The most commonly used format is a syringe barrel that contains a 20μm frit at the bottom of the syringe with the sorbent material and another frit on top, referred to as packed columns. Analytes can be classified into four categories; basic, acid, neutral and amphoteric compounds. Amphoteric analytes have both basic and acid functional groups and can therefore functions as cations, anions or zwitterions, depending on pH [17].

Figure.3-HPLC Instrument [25]

Gas Chromatography
GC The term Gas Chromatography (GC) is used for all methods when the mobile phase is gaseous. However, the stationary phase can be either solid (adsorption chromatography) or liquid fixed onto a solid carrier (partition or absorption chromatography). The sample can be either gas or liquid but in any case it is injected onto the column as gas or vapor. The consequence of this setup is that gas chromatography is suitable for the separation heat stable compounds i.e. chemicals which can be evaporated without decomposition. In GC the sample is injected into a continuous flow of the eluent through the injector. Then the sample is washed onto the column by the eluent where separation occur and finally components get into the detector ideally one after the other where a signal proportion to their concentration is generated. The whole system is controlled by a computer. Applied gases The source of the eluent i.e. mobile phase can be a high pressure cylinder or gas generator. The most often applied gases in a gas chromatography lab include He, Ar, N2 and H2. As GC is a high performance analytical method it is necessary to use high purity gases.

Depending on the purity of the gas (Table 1) different on-line gas purifiers should be installed, too.

Table 1: High purity gases

V/V % purity, gas content

Quality

Total impurities

99,99 %

4.0

100 ppm (v/v)

99,995 %

4.5

50 ppm (v/v)

99,999 %

5.0

10 ppm (v/v)

99,9995 %

5.5

5 ppm (v/v)

99,9999%

6.0

1 ppm (v/v)

Sample introduction
A critical point in gas chromatography is sample inlet. It is important to introduce the sample onto the column within the shortest possible time and at the same time to have it in the gas phase In theory, gas, liquid or even solid samples can be studied by GC but because of the slow evaporation of solids the samples injected are liquids or gases almost exclusively. Gas samples are injected by means of a six-port switching valve system. These injectors contain a sample loop of calibrated volume. First the loop is filled with the sample gas by washing it with the sample gas of 5-10 times volume This is necessary to be sure that the loop contains only the components of the sample and no air or remaining eluent gas. Upon switching, the content of the sample loop is washed into the carrier gas stream and injected to the column

Columns
The heart of the GC equipment is the column where the separation of sample components occurs. The column is placed into an oven with controlled temperature and computer controlled heating can rise its temperature up to 400-5000C but can be cooled to the initiate temperature also quickly. Columns can be divided into two groups. The so called packed columns are 1-5 m length and 2-6 mm internal diameter tubes filled with the appropriate stationary phase. As it has been mentioned already, in case of a separation based on adsorption the stationary phase could be a high surface area solid such as activated charcoal, Al2O3, silica, molecular sieve or organic polymer. In case of absorption based packed column the solid material is impregnated with the stationary liquid phase and then filled into the column.

Detectors
As it has been already discussed the components of the sample are separated on the column and get into the detector one after the other where a signal proportional with their concentration is generated by means of a physical method. The most often applied detectors in GC include Thermal Conductivity Detector (TCD), Flame Ionization Detector (FID), Electron Capture Detector (ECD), Mass Spectrometry (MS) detector and Infrared Spectrophotometric (IR) detectors. Other special detectors are also available such as Photoionization Detector (PID), Flame Photometric Detector (FPD), Pulsed Flame Photometric Detector (PFPD) or Atomic Emission Detector (AED).


Figure 4: GC Instrument[26]

CAPILLARY ELECTROPHORESIS-MASS SPECTROMETRY (CE-MS)
CE-MS, combining the high efficiency and resolution power of CE, with the high selectivity and sensitivity inherent to MS, is a very attractive analytical technique. However, CE-MS coupling, mostly by means of ESI [18], was not easy to implement since a closed electrical circuit is necessary not only for the electrophoretic separation but also for an efficient ionization in the source (with CE and ESI currents in the range of mA and nA, respectively). A solution for this problem is to ground the sprayer needle in order to divert all electrical energy from the CE to the ground and build an undisturbed electrical field for ionization in the MS source. Even if the sensitivity achieved with the use of a sheath flow is generally lower compared to sheath less interfaces, the robustness of the former system is generally better and detection limits in the low femtomole range can be achieved, especially when the flow rate of the sheath liquid is reduced to 500 nL/min. The detection of the narrow CE peaks requires the use of a fast and sensitive mass spectrometer. IT and TOF systems are adequate detectors because they acquire data over a suitable mass range with rates of several spectra per second.

CE-MS for bioanalysis of drugs
A number of recent reviews have covered the application of CE-MS for drug analysis, with some of them giving the fragmentations, when available, that the ionic species undergo in-source and in IT, triple quadrupole or TOF mass spectrometers [19]. This part of the review is dedicated to the analysis of drugs in biological fluids.

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