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5 - Liquid chromatography–mass spectrometry (LC–MS, or alternatively HPLC–MS)

5 - Liquid chromatography–mass spectrometry (LC–MS, or alternatively HPLC–MS)

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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

compounds.



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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.



17.5.4 INTERFACE

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).



17.5.5 APPLICATIONS

17.5.5.1 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. 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.



17.5.5.2 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 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

WISCONSIN–MADISON HTTP://UWLAB.SOILS.WISC.EDU)

17.6.1 INTRODUCTION

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

ICP-MS.



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

components.

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.



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CHAPTER 17  Analytical techniques



17.6.3 SAFETY

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.



17.6.4 INTERFERENCE

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

is recommended.

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

be interfered.



17.6.5  MEASUREMENT BY ICP-OES

17.6.5.1  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).



17.6.6 MEASUREMENT

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

17.6.7.1  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.



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