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Advanced laboratory technology

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Babla V Pendam, India Applications Manager, Agilent Technologies gives an outlook about various analytical techniques used in laboratories

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Babla V Pendam

The basic laboratory techniques complies to several GLP workflows involved for any analysis which is applicable to medicinal, chemical, microbiological, pharmaceutical and other laboratories. Although, in many of the cases, it could be limited to identifications, however, in the recent mutable scenarios, it is also demanding for the separation, quantitation and sometimes isolation as well.

To start with any of the experiments, it is always important for an analyst/ scientist to visualise the final goal so that the appropriate correct technique could be employed. In addition, the most advanced analytical techniques are now accessible, which could be very useful to meet the challenges as per the need and suiting the goals.

Apart from various other instrumental analytical techniques, liquid chromatography–mass spectrometry (LC-MS, or alternatively HPLC-MS) is the most important analytical chemistry technique that combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry (MS).

LC-MS is a powerful technique that has very high sensitivity and selectivity and so is useful in many applications. Its application is oriented towards the separation, general detection and potential identification of chemicals of particular masses in the presence of other chemicals (i.e., in complex mixtures), e.g., natural products from natural-products extracts and pure substances from mixtures of chemical intermediates. Preparative LC-MS systems can be used for rapid mass-directed purification of specific substances from such mixtures that are important in basic research, and pharma, agrochemical, food, and other industries.

Present-day liquid chromatography generally utilises very small particles packed and operating at relatively high pressure, and is referred to as high performance liquid chromatography (HPLC); modern LC-MS methods use HPLC instrumentation, essentially exclusively, for sample introduction. In HPLC, the sample is forced by a liquid at high pressure (the mobile phase) through a column that is packed with a stationary phase generally composed of irregularly or spherically shaped particles chosen or derivatised to accomplish particular types of separations.

HPLC methods are historically divided into two different sub-classes based on stationary phases and the corresponding required polarity of the mobile phase. Use of octadecylsilyl (C18) and related organic-modified particles as stationary phase with pure or pH-adjusted water-organic mixtures such as water-acetonitrile and water-methanol are used in techniques termed reversed phase liquid chromatography (RP-LC). Use of materials such as silica gel as stationary phase with neat or mixed organic mixtures are used in techniques termed normal phase liquid chromatography (NP-LC). RP-LC is most often used as the means to introduce samples into the MS, in LC-MS instrumentation.

MS is an analytical technique that measures the mass-to-charge ratio of charged particles. It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and other chemical compounds. MS works by ionising chemical compounds to generate charged molecules or molecule fragments and measuring their mass-to-charge ratios.

In a typical MS procedure a sample is loaded onto the MS instrument and undergoes vaporisation. The components of the sample are ionised by one of a variety of methods (e.g., by impacting them with an electron beam), which results in the formation of charged particles (ions).The ions are separated according to their mass-to-charge ratio in an analyser by electromagnetic fields. The ions are detected, usually by a quantitative method. Then the ion signal is processed into mass spectra.

Additionally, MS instruments consist of three modules:

  • An ion source, which can convert gas phase sample molecules into ions (or, in the case of electrospray ionisation, move ions that exist in solution into the gas phase).
  • A mass analyser, which sorts the ions by their masses by applying electromagnetic fields.
  • A detector, which measures the value of an indicator quantity and thus provides data for calculating the abundances of each ion present. The technique has both qualitative and quantitative uses. These include identifying unknown compounds, determining the isotopic composition of elements in a molecule, and determining the structure of a compound by observing its fragmentation. Other uses include quantifying the amount of a compound in a sample or studying the fundamentals of gas phase ion chemistry (the chemistry of ions and neutrals in a vacuum).

MS is now in very common use in analytical laboratories that study physical, chemical, or biological properties of a great variety of compounds.

LC-MS is very commonly used in pharmacokinetic studies of pharma and is thus the most frequently used technique in the field of bioanalysis. 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 is the use of tandem MS-MS. The detector can be programmed to select the ions to fragment. The selected ion then undergoes the fragmentation into product ions. This process is also known as multiple reaction monitoring which is very selective method. As long as there are no interferences or ion suppression, the LC separation can be quite quick. LCMS methods because of its selective properties can done in short runtimes as compared to over longer runs with UV detection. Since this methods are highly selective and can be much faster than traditional methods, this technique is preferred for studying drug metabolism and pharmacokinetic.

It is common now to have analysis times of one minute or less by MS-MS detection, compared to over 10 minutes with UV detection speed and throughput when using HPLC-MS/MS systems for drug metabolism and pharmacokinetic studies.

LC-MS is also used in proteomics, metabolonomics and more recent lipidomic studies where again components of a complex mixture must be detected and identified in some manner. The bottom-up proteomics 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 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.

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, quantitative bioanalysis and quality control.

MS is also used to determine the elemental composition and some aspect of the molecular structure of an analyte. Unique features of MS include its capacity for direct determination of the nominal mass of an analyte, and to produce and detect fragments of the molecule that correspond to discrete groups of atoms of different elements that reveal structure features.

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