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5 - Liquid chromatography–mass spectrometry (LC–MS, or alternatively HPLC–MS)
17.5 Liquid chromatography–mass spectrometry
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). 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.
17.5.1 FLOW SPLITTING
When standard bore (4.6 mm) columns are used the flow is often split ∼10:1. This
can be beneficial by allowing the use of other techniques in tandem such as MS
and UV detection. However, splitting the flow to UV will decrease the sensitivity of
spectrophotometric detectors. The mass spectrometry, on the other hand, will give
improved sensitivity at flow rates of 200 mL/min or less.
17.5.2 MASS SPECTROMETRY (MS)
Mass spectrometry (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 ionizing chemical compounds to generate charged molecules or molecule fragments and by measuring their mass-to-charge ratios. In a typical MS procedure, a sample is loaded onto the MS instrument and undergoes vaporization. The
components of the sample are ionized by one of a variety of methods (eg, 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 analyzer
by electromagnetic fields. The ions are detected, usually by a quantitative method.
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 ionization, move ions that exist in solution into the gas phase); a mass analyzer, which sorts
the ions by their masses by applying electromagnetic fields; and 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
CHAPTER 17 Analytical techniques
17.5.3 MASS ANALYZER
There are many different mass analyzers that can be used in LC/MS: single quadrupole, triple quadrupole, ion trap, time of flight (TOF), and quadrupole-time of flight.
Understandably, the interface between a liquid phase technique that continuously
flows liquid, and a gas phase technique carried out in a vacuum, was difficult for a
long time. The advent of electrospray ionization changed this. The interface is most
often an electrospray ion source or variant such as a nanospray source; however, atmospheric pressure chemical ionization interface is also used. Various deposition and
drying techniques have also been used such as using moving belts; however, the most
common of these is off-line MALDI deposition. A new approach still under development called Direct-EI LC-MS interface couples a nano HPLC system and an electron
ionization equipped mass spectrometer (Stobiecki et al., 2006).
LC–MS is very commonly used in pharmacokinetic studies of pharmaceuticals 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 ionized),
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.
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 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 such as 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.
17.6 Inductively coupled plasma spectrometry (ICP)
Profiling of secondary metabolites in plants or food like phenolics can be achieved
with liquid chromatography–mass spectrometry.
17.6 INDUCTIVELY COUPLED PLASMA SPECTROMETRY (ICP)
(SOIL & PLANT ANALYSIS LABORATORY UNIVERSITY OF
Inductively coupled plasma spectrometry is a type of mass spectrometry which is capable of detecting metals and several nonmetals at concentrations as low as one part
in 1015 (part per quadrillion, ppq) on noninterfered low-background isotopes. This
is achieved by ionizing the sample with inductively coupled plasma and then using
a mass spectrometer to separate and quantify those ions.
Compared to atomic absorption techniques, ICP has greater speed, precision, and
sensitivity. However, compared with other types of mass spectrometry, such as TIMS
and Glow Discharge, ICP introduces a lot of interfering species: argon from the plasma, component gasses of air that leak through the cone orifices, and contamination
from glassware and the cones.
Analysis of major, minor, and trace elements in plant tissue samples can be done
by inductively coupled plasma optical emission spectrometry (ICP-OES) and inductively coupled plasma mass spectrometry (ICP-MS). It allows determination of elements with atomic mass ranges 7–250 (lithium to uranium), and sometimes higher.
ICP is also used widely in the geochemistry for radiometric dating, in which it is
used to analyze relative abundance of different isotopes, in particular uranium and
lead. In the pharmaceutical industry, ICP is used for detecting inorganic impurities
in pharmaceuticals and their ingredients. One of the largest volume uses for ICP is
in the medical and forensic field, specifically, toxicology. In recent years, industrial
and biological monitoring has presented another major need for metal analysis via
17.6.2 SUMMARY OF METHOD
Half a gram of dried sample (or equivalent) and 5 mL of concentrated nitric acid
are added to a 50-mL Folin digestion tube. The mixture is heated to 120–130°C for
14–16 h and is then treated with hydrogen peroxide. After digestion, the sample
is diluted to 50 mL. This solution is analyzed by ICP-OES for major and minor
components, and further 1:1 diluted and analyzed by ICP-MS for minor and trace
After solid samples are converted into solution samples, the procedures of “elemental analysis of solution samples with ICP-OES” and “elemental analysis of solution samples with ICP-MS” are followed.
CHAPTER 17 Analytical techniques
All chemicals should be considered potential health hazard. All relevant laboratory
safety procedures are followed.
The use of perchloric acid for a sample digestion must be conducted in a hood
designed specifically for perchloric acid. The user must be aware of the dangers involved using perchloric acid, such as the explosive nature of anhydrated perchloric
acid and its extreme corrosive nature.
This method covers the analysis of over 30 elements in different kinds of samples by
ICP-OES and ICP-MS. A general discussion of interference is lengthy but not necessarily relevant to a specific element, which is especially true if the sample matrix is
not specifically defined. An enormous amount of literature is available to the analysis
of metals and nonmetals by ICP-OES and by ICP-MS. Reading the published articles
In this method, the solution contains less than 1000 ppm of dissolved solids for
ICP-OES and ICP-MS analysis. The major components are K, Mg, Ca, P, S, and Na.
These components either do not pose significant interferences with other elements/
isotopes or the potential interferences are well understood and controlled. Significant
interferences are not expected, although some specific elements and or isotopes may
17.6.5 MEASUREMENT BY ICP-OES
22.214.171.124 Sample preparation
Set 8-mL autosampler tubes in ICPOES sample racks. Transfer sample solutions
from 50-mL tubes to 8-mL tubes. For samples with extremely high analytes, the
samples may be further diluted. Add 3 mL of sample solution and 3 mL of 2% nitric acid to the 8-mL autosampler tube (2nd dilution. Nominal dilution factor = 200.
Y = 4 ppm).
A detailed procedure is given in “elemental analysis of solution samples with ICPOES.” Digestion blanks are also measured with other samples.
17.6.7 MEASUREMENT BY ICP-MS
126.96.36.199 Sample preparation
Set 14-mL Falcon tubes in the ICPMS autosampler racks. Transfer the sample solutions to the Falcon tubes. Adjust the volume to 5 mL. Add 5 mL of 2% nitric acid.
Mix well. The nominal dilution factor is 200 and the IRS is 4 ppb of Rh. Since an internal reference standard is used, the volume inaccuracy during dilution is i rrelevant.