A CRITICAL REVIEW ON PHARMACEUTICAL ANALYSIS IN LIQUID CHROMATOGRAPHY - MASS SPECTROSCOPY (LCMS)

About Authors:
P. Udaya Lakshmi*, B.Krishnamoorthy, M.Muthukumaran, Amreen Nishat
Montessori Siva Sivani Institute of Science&Technology College of Pharmacy-Mylavaram,
Vijayawada, Andhrapradesh-521230
*udayalakshmi53@gmail.com

ABSTRACT
Liquid chromatography (LC) combined with mass spectrometry (MS) is a powerful tool for qualitative and quantitative analytics of organic molecules from various matrices, and the use of this hyphenated technique is very common in bioanalytical laboratories. LC-MS is an analytical technique that couples high resolution chromatographic separation with sensitive and specific mass spectrometric detection. The technique is still fast developing, particularly in the mass spectrometry area, with vastly improved sensitivity and resolution. It is probably the most powerful technique currently available for pharmaceutical analysis. The role of LC/MS in the pharmaceutical industry during the past decade is examined, and key elements for recent success are illustrated to include significant advances in instrumentation, methodology, and application. The applications are highlighted with reference to the analysis opportunity and analysis strategy implemented. The applications of LC-MS to the studies of in vitro and in vivo drug metabolism, identification and characterization of impurities in pharmaceuticals, analysis of chiral impurities in drug substances and high-throughput LC-MS-MS systems for applications in the "accelerated drug discovery" process are described.

REFERENCE ID: PHARMATUTOR-ART-1661

INTRODUCTION
Coupling of MS to chromatographic techniqueshas always been desirable due to the sensitive and highly specific nature of MS compared to other chromatographic detectors. The coupling of MS with LC (LC-MS) was an obvious extension but progress in this area was limited for many years due to the relative incompatibility of existing MS ion sources with a continuous liquid stream. Several interfaces were developed but they were cumbersome to use and unreliable, so uptake by clinical laboratories was very limited. This situation changed with the development of the electrospray ion source by Fenn in the 1980s ^[1].

Major advantages of LCMS are as the follows:
·         It delivers high-quality, fully library matchable mass spectra of most sub-1 kDa molecules amenable by HPLC.
·         It is a chemical ionisation free interface (unless operated intentionally) with accurate reproduction of the expected isotope ion abundances.
·         Response is never influenced by matrix components in the sample or in the mobile phase.

It can be considered a universal detector for small molecules because response is not related to compound polarity.

DEVELOPMENTS IN LC-MS
Brief History of LC-MS The last twenty years has seen a dramatic increase in the capabilities of MS. At the beginning of this period the invention of fast atom bombardment (FAB), by Barber et al. in 1981, ^[2] enabled easier analysis of involatile and thermally unstable molecules, especially those of biological interest. It may be argued that this technique acted as a catalyst for the development of other ionization techniques, such as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), applicable to such molecules. The combination of liquid chromatographic techniques with MS has been an important development. Early interfaces were concerned with coping with the potential of 1 l/min vapour in the MS ionisation source vacuum that would be generated by eluent from typical analytical columns flowing at 1 ml/min. This was achieved by evaporating solvent on a heated moving belt ^{[3, 4]} or the use of very low flows, such as direct liquid introduction ^[5,6] and continuous flow FAB (CF-FAB).^[7]Thermospray ^[8] and particle beam ^[9] interfaces were improved methods during the 1980s.

WHY LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY?
Liquid chromatography is a fundamental separation technique in the life sciences and related fields of chemistry. Unlike gas chromatography, which is unsuitable for nonvolatile and thermally fragile molecules, liquid chromatography can safely separate a very wide range of organic compounds, from small-molecule drug metabolites to peptides and proteins.

Traditional detectors for liquid chromatography include refractive index, electrochemical, fluorescence, and ultraviolet-visible (UV-Vis) detectors. Some of these generate two- dimensional data; that is, data representing signal strength as a function of time. Others, including fluorescence and diode- array UV-Vis detectors, generate three-dimensional data. Three-dimensional data include not only signal strength but spectral data for each point in time.

Mass spectrometers also generate three- dimensional data. In addition to signal strength, they generate mass spectral data that can provide valuable information about the molecular weight, structure, identity, quantity, and purity of a sample. Mass spectral data add specificity that increases confidence in the results of both qualitative and quantitative analyses.

For most compounds, a mass spectrometer is more sensitive and far more specific than all other LC detectors ^[10]. It can analyze compounds that lack a suitable chromophore. It can also identify components in unresolved chromatographic peaks, reducing the need for perfect chromatography. Mass spectral data complements data from other LC detectors. While two compounds may have similar UV spectra or similar mass spectra, it is uncommon for them to have both. The two orthogonal sets of data can be used to confidently identify, confirm, and quantify compounds.

Some mass spectrometers have the ability to perform multiple steps of mass spectrometry on a single sample. They can generate a mass spectrum, select a specific ion from that spectrum, fragment the ion, and generate another mass spectrum; repeating the entire cycle many times. Such mass spectrometers can literally deconstruct a complex molecule piece by piece until its structure is determined.

MECHANISM
The interfacing mechanism is contained inside a common EI source, like that found in any GC-MS system. The liquid phase from a nano HPLC column is admitted from the capillary column port, where the connection tubing and the nebulizer are first introduced and sealed to prevent vacuum loss. The mechanism is based on the formation of an aerosol in high-vacuum conditions, followed by a quick droplet desolvation and final vaporization of the solute prior to the ionization. The completion of the process is quick and complete and reduces chances of thermal decomposition as reported in the Figure no:1, where a scheme of the interface is shown. The core of the interface is represented by the micro-nebulizer. The nebulizer tip protrudes into the ion source so that the spray expansion is completely contained inside the ion volume. The eluate emerges as liquid phase at a flow rate of 300-500 nL/min, and any premature in-tube solvent evaporation is prevented by a convenient thermal insulation of the nebulizer and the connecting tubing from the surrounding source heat. The high temperature of the ion source, between 300 and 400°C, has a double function: to compensate for the latent heat of vaporization during the droplet desolvation, and to convert the solute into the gas phase. If all components of this simple interface are correctly placed and sized, then each substance separated by the nano-column is smoothly converted into the gas phase, the peak profile is nicely reproduced, and high quality mass spectra are generated^[11].

MASS SPECTROMETRY INSTRUMENTATION
Mass spectrometers operate by converting the analyte molecules to a charged (ionised) state, with subsequent analysis of the ions and any fragment ions that are produced during the ionisation process, on the basis of their mass to charge ratio (m/z). Several different technologies are available for both ionisation and ion analysis, resulting in many different types of mass spectrometers with different combinations of these two processes. In practice, some configurations are far more versatile than others and the following descriptions focus on the major types of ion sources and mass analysers likely to be used in LC-MS systems within clinical laboratories.

1) ION SOURCE
1.1) Electrospray Ionisation Source
Fenn developed ESI into a robust ion source capable of interfacing to LC and demonstrated its application to a number of important classes of biological molecules. Liquid samples are pumped through a metal capillary maintained at 3 to 5 kV and nebulised at the tip of the capillary to form a fine spray of charged droplets. The capillary is usually orthogonal to, or off-axis from, the entrance to the mass spectrometer in order to minimise contamination. The droplets are rapidly evaporated by the application of heat and dry nitrogen, and the residual electrical charge on the droplets is transferred to the analytes. The ionised analytes are then transferred into the high vacuum of the mass spectrometer via a series of small apertures and focusing voltages. The ion source and subsequent ion optics can be operated to detect positive or negative ions, and switching between these two modes within an analytical run can be performed.

1.2) Atmospheric Pressure Chemical Ionization Source
In atmospheric pressure chemical ionisation (APCI), as with ESI, liquid is pumped through a capillary and nebulised at the tip. A corona discharge takes place near the tip of the capillary, initially ionising gas and solvent molecules present in the ion source. These ions then react with the analyte and ionise it via charge transfer. The technique is useful for small, thermally stable molecules that are not well ionised by ESI ^[12]. For example, free steroids do not ionise well using ESI because they are neutral and relatively non-polar molecules, lacking a functional group capable of carrying charge. APCI has therefore been used to improve the sensitivity of LC-MS analysis of free steroids.

1.3) Atmospheric Pressure Photo-ionisation
Atmospheric pressure photo-ionisation (APPI) uses photons to excite and ionise molecules after nebulisation. The energy of the photons is chosen to minimise concurrent ionisation of solvents and ion source gases. The technique also gives predominantly singly-charged ions ^[13] and has been used for the analysis of neutral compounds such as steroids^.

1.4) MALDI (matrix assisted laser desorption ionization)
The matrixes used are sinapinic acid and 2, 5 di hydro benzoic acid. Analyte is mixed with large excess of matrix and irradiated by short pulse of laser light. It is used for analysis of biomolecules and large organic molecules. It involves 2 steps i.e desorption by laser beam and ionization_[14]

2. MASS ANALYZERS
1. Single focusing mass analyzer
It involves magnetic field, causes ions to be deflected along curved paths. Intially set of electrostatic slits acclerates the ions from source forms them into narrow beam and directs them towards magnetic field. Velocity of ions depends on potential V applied on slits. The ions travel in a path perpendicular to the magnetic field applied. Magnetic field, H, separates the ions a/c to m/z ratio. Ions of single m/z value will move in a trajectory path. By changing the magnetic field strength, ions of ring m/z values are brought to focus of detector slit. so agnetic field classifies and segregates ions into beams each with different m/z ratio.To obtain mass spectrum, either accelerating voltage (or) magnetic field is varied.

m/z = h²r²/2V

2. Double focussing sector analyzer
Magnetic field and electric field are used to disperse ions according to their momentum and translational energy. Electrostatic deflection is the field incorporation between ion source and mass analyzers. As ions pass through electrostatic sector, dispressed according to translational energy. Only ions with correct translational energy pass the electrostatic sector. Magnetic sector disperses according to momentum. a mass spectrum is obtained by scanning the magnetic field strength to bring ions with different m/z ratios^[15] focus at detector.

3. Quadrapole mass analyzer
It consists of 4 parallel circular rods. Ions are separated based on stability of their trajectories in oscilladting electric fields that are applied between one pair of rods and the other. a direct current voltage is superimposed on RF voltage. Ions travel down quadrapole between rods. Only ions of certain m/z ratio will reach detector for given ratio of voltages, others have unstable trajectories and will collide with the rods. this permitts selection of ions with particular m/z (or) allows operator to scan for range of m/z by varying applied voltage.Quadrupole only analyzers show much lower sensitivity^[16] because they scan (as do magnetic sector analyzers) and spend very little time at any particular m/z value.

4. Time of flight analyzer (TOF)
The resurgence of interest in TOF analysers probably arose from their use in MALDI. It is based on kinetic energy and velocity of ions. All the ions have same kinetic energy. So, the time to reach the detector travel along the tube depends on mass. Ions have same energy; velocity dependent velocity is inversly proportional to (m)^1/2. So, ions with high velocity (low m/z) reach faster.

Time of flight (t_f)=L.[(m/z)(1/2V)]^1/2

APPLICATIONS

1. Pharmacokinetics
LC-MS is very commonly used in pharmacokinetic studies of pharmaceuticals and is thus the most frequently used technique in the field of bioanalysis ^[17]. These studies give information about how quickly a drug will be cleared from the hepatic blood flow, and organs of the body. MS is used for this due to high sensitivity and exceptional specificity compared to UV (as long as the analyte can be suitably ionised), and short analysis time.

The major advantage MS has is the use of tandem MS-MS. The detector may be programmed to select certain ions to fragment. The process is essentially a selection technique, but is in fact more complex. The measured quantity is the sum of molecule fragments chosen by the operator. As long as there are no interferences or ion suppression, the LC separation can be quite quick. It is common now to have analysis times of 1 minute or less by MS-MS detection, compared to over 10 mins with UV detection.

2. Proteomics/metabolomics
LC-MS is also used in proteomics where again components of a complex mixture must be detected and identified in some manner. The bottom-up poteomics LC-MS approach to proteomics generally involves protease digestion and denaturation (usually trypsin as a protease, urea to denature tertiary structure and iodoacetamide to cap cysteine residues) followed by LC-MS with peptide mass fingerprinting or LC-MS/MS (tandem MS) to derive sequence of individual peptides. LC-MS/MS is most commonly used for proteomic analysis of complex samples where peptide masses may overlap even with a high-resolution mass spectrometer. Samples of complex biological fluids^[18] like human serum may be run in a modern LC-MS/MS system and result in over 1000 proteins being identified, provided that the sample was first separated on an SDS-PAGE gel or HPLC-SCX. Profiling of secondary metabolites in plants or food like phenolics can be achieved with liquid chromatography–mass spectrometry.

3. Drug development
LC-MS is frequently used in drug development at many different stages including Peptide Mapping, Glycoprotein Mapping, Natural Products Dereplication, Bioaffinity Screening, In Vivo Drug Screening, Metabolic Stability Screening, Metabolite Identification, Impurity Identification, Degradant Identification, Quantitative Bioanalysis, and Quality Control ^[19].

(a) In Vitro Drug Metabolism
The liver is the primary organ that metabolizes drugs. Preliminary studies of drug metabolism have therefore commonly been carried out in vitro with liver microsomal preparations or hepatocytes^[20—22] These provide good initial indication of the metabolic fate of a drug. The metabolism of droloxifene, ^[23] an analogue of the antibreast cancer drug tamoxifen, by human liver microsomes is an example of the application of LC-MS in an in vitro metabolism study.

(b) In Vivo Drug Metabolism
In vivo metabolism studies involve analysis of drugs and metabolites in blood, urine and faeces. These samples contain a larger amount of endogenous compounds that could co-elute and interfere with the LC-MS analysis. A good sample preparation technique coupled with efficient chromatographic separation is therefore essential for the successful application of LC-MS to in vivo metabolism studies.

4. Other Applications
Direct-EI may offer a clear advantage over ESI in several applications, but it may excel in the following cases:
· Large number of compounds of different polarities and chemical properties: EI can offer a shortcut, do-it-all solution when hard-to-detect substances are included or and when a combination of positive and negative ion detection runs are required for complete coverage of analyte detection.

· Characterization of unknowns: library matching offer an invaluable tool for compound identification.

· Detection of non chromophoric compounds that also give poor or no signal with API: for these compounds additional HPLC detectors such as evaporative light scattering detector (ELSD), refractive index (RI) or corona discharge aerosol detector (CAD) are also available but each of them has limitations which restrain obtaining a universal detection with reasonable sensitivity. EI-MS would offer a suitable solution for this type of compounds, in terms of sensitivity and universal response. GC is anyway feasible only for compounds with high to medium volatility and therefore cannot be adopted for a full characterization of mixtures of complex nature ^[24]. The possibility of hyphenating EI to HPLC separation represents an ideal solution.

· Quantitative analyses in presence of matrix effects: EI-MS offers a superior performance compared to ESI or APCI when intruding interferences from complex matrices pass clean up procedure and cause signal suppression or enhancement

CONCLUSION
LCMS have proved to be a very valuable tool for the analysis of non-volatile high molecular weight and thermolabile compounds. MS alone is not sufficient to give adequate information hence LCMS /MS or tandem MS is used which have more sensitivity, selectivity, resolution and gives more information about structure. LC/MS/MS enhances efficiency in workflow for simultaneous multi component analysis enabling researches start their analysis with out setting complicated separation parameter or optimizing the MS parameter to each compound. Thus LCMS/MS found a place in every Drug development activity right from research to toxicology studies.

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