LCMS Introduction Part 2

8/2/2019 LCMS Introduction Part 2

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BioAnalytical Technologies (India) Pvt. Ltd.

BASICS OF LCMS(VOLUME -II)

BioAnalytical Technologies (India)

Plot No. EL-72, Electronic Zone,

TTC Industrial Area, Mahape,

Navi Mumbai - 400 705


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TABLE OF CONTENTS

10. METHOD OPTIMIZATION FOR LCMS ........................................................3

11. DATA INTERPRETATION..............................................................................10

12. APPLICATIONS OF LCMS .............................................................................18

13. SOFTWARE- ANALYST ..................................................................................22

14. REFERENCES....................................................................................................32

15. FURTHER STUDY.............................................................................................33


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LIST OF FIGURES

FIGURE 10.1ISOCRATIC SEPARATION:EFFECT OF COLUMN DIAMETER.................................................................................6 FIGURE 10.2ISOCRATIC SEPARATION:EFFECT OF COLUMN VOLUME ...................................................................................6 FIGURE 10.3ISOCRATIC SEPARATION:EFFECT OF COLUMN LENGTH. ................................................................................... 6FIGURE 10.4ISOCRATIC SEPARATION:EFFECT OF FLOW RATE..............................................................................................7 FIGURE 10.5GRADIENT SEPARATION:GRADIENT WASTE .....................................................................................................8 FIGURE 10.6GRADIENT SEPARATION: COLUMN LENGTH.......................................................................................................8 FIGURE11.1TYPICAL MASS SPECTRUM ...............................................................................................................................10 FIGURE 11.3CLEAVAGE FRAGMENTATION OF AMINES. ............................................................................................ ...........14FIGURE 11.5MCLAFFERTY REARRANGEMENT MECHANISM. ............................................................................................... 15FIGURE 11.6ISOTOPIC ABUNDANCE -70 EVEI OF CH3CL.................................................................................................16 FIGURE 11.7ISOTOPIC ABUNDANCE-70 EVEI OF CH2CL2................................................................................................16 FIGURE 13.1:SECURITY........................................................................................................................................................24 FIGURE 13.2:MANUAL TUNING ...........................................................................................................................................25 FIGURE 13.3A:ACQUIREDATA ACQUISITION METHOD EDITOR......................................................................................26

FIGURE 13.3B:ACQUIREDATA ACQUISITION BATCH EDITOR..........................................................................................27 FIGURE 13.4:EXPLORING DATA ...........................................................................................................................................28 FIGURE 13.5A:QUANTITATIVECREATE QUANTITATION METHOD ...................................................................................29


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10. Method Optimization for LCMS

Simplifying HPLC methods to LCMS

Introduction

This topic gives a brief knowledge about the parameters to be considered for sample preparation in

Liquid Chromatography Mass Spectrometer.

LC-MS is a technique that combines the solute separation power of HPLC, with the exquisite

detection power of a mass spectrometer. HPLC is also an excellent way to remove potentially

interfering molecules from the sample such as salts, buffers and detergents. These types of moleculesgreatly influence the efficiency of the ionization and the quality (and quantity) of data generated by

the MS is greatly dependent on a clean sample prior to ionization. Coupling a high performanceliquid chromatography (HPLC) system with a mass spectrometer has proved to be a difficult task anda great deal of research has gone into this problem. The difficulty has been that the HPLC system

deals with analyte in the liquid-phase yet the MS requires a transformation of these ions from the

liquid phase to ions in the gas. It is challenging to maintain a sufficient vacuum level in the mass

spectrometer because introduction of a liquid at the ion source wreaks havoc on the vacuum. For this

reason the solvent must be stripped and gas phase ions must be generated before introduction to theMS.

This topic looks at some of the important factors to consider when converting conventional HPLC

methods to methods run on the mass spectrometer.

Glossary

1. Capacity Factor (k): Chromatographic parameter, which specifies the degree ofretention delay of a substance to be separated, called as Retention Factor.

2. Column Volume: The geometric volume of the part of the tube that contains thepacking.

3. Dwell Volume: The dwell volume is the volume from the point the solvent ismixed until it hits the head of the column.

4. Elution: It is transport of the substance to be separated using the mobile phasethought the column.


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5. Gradient Separation: A technique for decreasing separation time by increasingstrength of a mobile phase over time interval during chromatographic separation.

The gradient can be continuous or step wise.

6. Isocratic Separations: Elution of a substance/mixture at constant mobile phasestrength or composition.

Steps Converting LC to LCMS Methods

One big challenge in coupling LC methods to MS detection occurs at the interface.

Mobile phase leaves the LC in liquid form and must be converted to the gas phase approximately a

1000-fold increase in volume. This requires that the mobile phase, often with a high percent of water, be vaporized thoroughly and quickly. Any additives to the mobile phase, such as buffers or ion

pairing reagents, must also be volatile. A small portion of this vaporized LC column effluent is then

transferred into the MS for detection.

The conversion of LC methods can be thought of in three steps.

1. Converting LC mode to MS- compatible mobile phase.

2. Adjusting column conditions with respect to MS.

3. Confirming the MS methods.

First, the mobile phase may need adjustment for compatibility. This often is best done on the LC

platform where the method is already running. There is no need for the added complication of MS

detection or the cost of tying up a more expensive detector at this stage. This means shorter columns,shorter run times, and less resolution. This can be done on either platform. Finally, the method is

moved to the LC/MS and any final adjustments can be made.

The choice of mobile phase when developing an LC method destined for MS detection can influencethe compatibility of the two techniques. The mobile phase must be sufficiently volatile to vaporize

completely in the interface. Modern LC/MS interfaces are capable of vaporizing high water content

mobile phases, but all other things being equal, higher organic phases are preferred The choice of

solvent also can impact sample ionization, and acetonitrile usually is the first choice. Often more

important than the solvents is the choice of buffers. Phosphate is the most popular LC buffer, but it isnot sufficiently volatile for routine LC/MS use. Generally lower concentrations of buffers are used

for LC/MS than for LC. This means a balance needs to be met between having sufficient buffering

capacity while keeping the total ion content as low as possible. The most effective LC/MS buffersare formic acid, ammonium acetate, ammonium formate and trifluroacetic acid. Many times all that

is needed is a low pH without much buffering capacity. So formic acid or sometimes acetic acid can

be used at 0.1% in the mobile phase to both adjust the mobile phase pH and to aid performance of

the MS. Higher pH values can be obtained with ammonium acetate or ammonium formate. AlthoughTFA is volatile and effective for low pH work, it is much more likely to suppress ionization in the

MS than formic acid, so it is seldom the best additive to use.

Once an MS-compatible mobile phase has been identified, the next step is to scale the column to take

maximum advantage of the MS. This can be done on the LC with a conventional detector or on the

LC/MS directly, depending on instrument availability. Or isocratic separation, it generally is

desirable to have retention factors in the 1 < k < 20 region for good chromatography. Theseguidelines need some modification for LC/MS. Selectivity is defined as the ratio of k-values for

adjacent peaks, so in order to maintain selectivity, or relative peak spacing, one needs to keep kconstant in isocratic separation. With LC methods, one usually wants sufficient retention so that the

garbage at the solvent front doesnt interfere with the analyte. This usually means k > 1. With

LC/MS, this early-eluting material can suppress ionization of analytes. Suppressed ionization canlead to non-linearity and inaccurate quantification. One easy way to check for suppression is shown

here. A constant concentration of a standard is infused into the mobile phase stream after the column.

Once the baseline stabilizes, an injection of an extracted matrix blank is made. At the solvent front a

negative dip will be seen as ionization-suppressing materials elute and reduce the baseline signal.When the baseline returns to normal, all these suppressing agents have passed through the detector.

Chromatographic conditions should be adjusted so that the peaks of interest elute after this time.


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As the separation is scaled from LC to LC/MS, the chromatography column should be used to its

maximum advantage. Because MS reduces the chromatographic resolution requirements and because

run time often is reduced to improve sample throughput, shorter columns often are used with LC/MSapplications. Care needs to be taken when scaling down traditional LC methods for LC/MS use.

Mobile Phase Requirement

Mobile phase degassing is an important step in the LC/MS experiment and can be accomplished viaon-line membrane or vacuum devices, sonication, helium sparging. Degassing will eliminate pump,

cavitation, ensure reproducible retention times and minimize possible sputtering from ion the source.Acetonitrile and Methanol are almost exclusively chosen in LC/MS method as organic mobile-phase

components. Methanol has greater phase acidity, polarity, and volatility than acetonitrile. It has been

shown 10-50% better sensitivity than acetonitrile in positive ion mode.

The choice of mobile phase when developing an LC method destined for MS detection can influencethe compatibility of the two techniques. The mobile phase must be sufficiently volatile to vaporize

completely in the interface. Modern LC/MS interfaces are capable of vaporizing high water content

mobile phases, but all other things being equal, higher organic phases are preferred The choice ofsolvent also can impact sample ionization, and acetonitrile usually is the first choice.

Often, more important than the solvents is the choice of buffers. Phosphate is the most popular LC

buffer, but it is not sufficiently volatile for routine LC/MS use. Generally lower concentrations of buffers are used for LC/MS than for LC. This means a balance needs to be met between having

sufficient buffering capacity while keeping the total ion content as low as possible. Many times all

that is needed is a low pH without much buffering capacity. So formic acid or sometimes acetic acidcan be used at 0.1% in the mobile phase to both adjust the mobile phase pH and to aid performance

of the MS. Higher pH values can be obtained with ammonium acetate or ammonium formate.

Although TFA is volatile and effective for low pH work, it is much more likely to suppressionization in the MS than formic acid, so it is seldom the best additive to use.

Isocratic Separation

When scaling isocratic separations, the use of column back-pressure can be used as a guide to

adjustment of conditions. One should retain the same linear velocity of solvent to obtain similar

column efficiency. Some of the important parameters, which need to be standardized in isocraticseparation:

Adjust column diameter:

Reduction of the column diameter from 4.6 mm to 2.1 mm requires a 5-fold reduction in flow rate to

obtain constant velocity and constant pressure. Under these conditions, one would expect the

chromatographic separation to be unchanged. In this example, the column diameter was reduced

from 4.6 mm to 2.1 mm, requiring a 5-fold reduction in flow rate to maintain the same pressure andlinear velocity. Note that the retention times are not exactly the same because the ratio of column

diameter squared is not exactly 5. Note also that although the peak spacing is not changed, theseparation has degraded in the second case. This is because of extra-column effects (figure 10.1).


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Figure 10.1 Isocratic Separation: Effect of column diameter

Eliminate extra column volume:

Extra-column volume contributes to band spreading (Fig 10.2). This generally is not noticeable with

4.6 mm columns 150-250 mm long, as used with conventional LC. However, when column volume

is reduce, as with going to a 2.1 mm i.d. column, extra-column volume can be significant. In thiscase, extra-column volume was reduced by replumbing the system with short lengths of 0.005 i.d.

tubing. Now the separations are nearly identical, as expected.

Figure 10.2 Isocratic Separation: Effect of column volume

Reduce column length:

Because MS requires much less chromatographic resolution than conventional LC separations, theseparation in the top run is excessive, wasting time and increasing analysis cost. In this case, the

column was replaced with a shorter column, with the expected 3-fold drop in retention and pressure

plus reduced resolution (Figure 10.3).

Figure 10.3 Isocratic Separation: Effect of column length.


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Increase flow rate:

The shorter column reduced the system pressure. Generally pressures of 1500- 2500 psi are

acceptable, so the flow rate was increased by a factor of three to produce the lower separation. Small-

particle columns are not highly sensitive to flow rate, so the lower separation is not much worse thanthe upper one (Figure 10.4).

Figure 10.4 Isocratic Separation: Effect of flow rate

By reducing column diameter, column length and flow rate, the original LC method taking about 10

min now is suitable for LC/MS use, with a cycle time of


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Figure 10.5 Gradient Separation: Gradient waste

Shortening the column:

A shorter column is used. The 5-fold change in length requires a 5-fold reduction in the gradient time

to keep k* constant. In addition to shortening the column, the particle size was reduced to 3 microns(Figure 10.6). Some minor changes in the separation are noticed; these will be addressed a little later.

Figure 10.6 Gradient Separation: column length.

Reduce column diameter:

The column diameter is changed from 4.6 mm to 2.1 mm. As with the isocratic case, this require a 5-

fold reduction in flow rate to keep the velocity constant and to keep k* constant. The reduction in

column diameter didnt have the expected effect. Instead of similar resolution and retention, reducedresolution and increased retention are observed. This problem is due to the gradient dwell volume.

The dwell volume is the volume from the point the solvent is mixed until it hits the head of the

column. This includes the mixer, plumbing, and injector loop. For high pressure mixing systems,

dwell volumes tend to be in the 0.1-3 mL range, whereas low pressure mixing systems typically have2-5 mL dwell volumes. The dwell volume is important especially when scaling or transferring a

method. In this case, reduction of the column diameter required a 5-fold lower flow rate. But insteadof equivalent separation, the run time is considerably longer. When the gradient profile is overlaid onthe separations, the cause is obvious. The isocratic section of the gradient results from the time it

takes for the gradient to flow through the dwell volume from the mixer to the head of the column.

With a dwell volume, VD, of 1.0 mL, this is 1 min in the top run, but 5 min in the lower one. This

additional isocratic hold is responsible for the observed changes in retention and selectivity. Ration,

particularly of early peaks, is changed. Just as it was necessary to take care to keep k constant whenscaling the column parameters, one must scale the dwell volume in accordance with the flow rate to

obtain similar dwell times. In practice, one typically replaces the standard LC mixer with a

micromixer when using LC/MS. Thus, a 2.5 mL mixer might be replaced with a 10microlitermicromixer. Once the dwell volume is reduced, the same separation is obtained with both conditions.


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If a smaller dwell volume were used, one could add a short isocratic hold to the method to lengthen

the isocratic hold to 1 min total.

Ballistic Gradients:

Ballistic gradient is often used for LC/MS2. These are referred to with a smirk, suggesting the

chromatography is terrible. Although the conditions look extreme, calculation of k indicates that thestated conditions do produce good chromatography. For good chromatographic performance, one

generally wants k to be in the region of 2 to 10.

Ballistic gradients are not inherently bad, but there are some pitfalls to be careful of. Calculation of k

is essential to obtain good chromatographic properties. The equipment should be checked to be sure

it could indeed generate such short, steep gradients. Some equipment will produce curved, not lineargradients under such conditions. The dwell time can be very important whenever small columns are

used. Be sure to allow sufficient retention so that the early-eluting material does not suppress

ionization and that the system produces sufficient resolution for the MS.

Summary

One should be sure to use a mobile phase and buffer that are both volatile and support ionization of

the sample compounds. When scaling isocratic runs, adjust the flow rate as the column diameter is

changed so that the pressure and linear velocity stay the same. The column length can be reduced soas to give up extra resolution and reduce run time. Scaling gradients is a little more complex, but if

conditions are adjusted to keep k constant, the procedure is quite logical. One must also take care to

avoid problems due to extra-column effects and excessive dwell volume. Finally, ballistic gradients

are OK to use, but calculations of k must be done to ensure the best possible chromatogram is

obtained even though the run is short.

2A ballistic gradient is a very fast separation technique used mostly in LC-MS applications; the

complete analysis can take less than one minute and up to five. Non-optimal, high flow rates orlinear velocity are combined with fast gradients and very short columns.


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11. DATA INTERPRETATION

Indulging the spectrum

INTRODUCTION

The ions which get detected in Mass Spectrometer are is displayed in form of a mass spectra. Most

mass spectrometers ionize the particles, or break the particles into fragments whose relativeabundance is measured; others observe the intact molecular masses without fragmentation.

Figure11.1 Typical Mass Spectrum

A Mass spectrum of a sample is a pattern representing the distribution of atoms or molecules by

mass in the substance. A mass spectrum will usually be presented as a vertical bar graph, in whicheach bar represents an ion having a specific mass-to-charge ratio (m/z) and the length of the bar

indicates the relative abundance of the ion (figure 11.1). The most intense ion is assigned an

abundance of 100, and it is referred to as the base peak. The X-axis of a mass spectrum is a mass-to-

charge ratio. The y-axis of a mass spectrum represents relative abundance of particles, and has

arbitrary units.

Most of the ions formed in a mass spectrometer have a single charge, so the m/z value is equivalentto mass itself. Modern mass spectrometers easily distinguish (resolve) ions differing by only a single

atomic mass unit (amu), and thus provide completely accurate values for the molecular mass of acompound. The highest-mass ion in a spectrum is normally considered to be the molecular ion, andlower-mass ions are fragments from the molecular ion, assuming the sample is a single pure

compound.

The index field collects index entries specified by XE. To insert an index entry field, select the text

to be indexed, and choose Index and Tables from the Insert menu. Click on the Index tab to receivethe Index dialog box.


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Glossary

1. Accurate Mass :Isotopes have unique precise masses, a consequence of which isthat the elemental composition of any molecule, or fragment of one, can be

calculated from its mass if this is sufficiently accurately determined.

2. Average Mass (Mr): The mass of a particle or molecule of given empiricalformula calculated using atomic weights for each element.

3. Base Peak: The most intense ion detected in the spectrum.

4. Isotopes: Most elements are composed of a mixture of isotopes. These will beseparated in a mass spectrometer. Atoms or molecules containing such elements

will display a cluster of ions reflecting the isotopic composition.

5. Molecular Ion: The ion formed from the original molecule in the source.

6. Monoisotopic Ion: The ion containing only the most abundant isotopes. (alsocalled the principal ion).

7. Monoisotopic Ion Mass: The mass of an ion containing the most abundantisotopes, calculated with exact atomic weights.

8. m/z :The ratio of charge to mass of the ion detected. z is often unity but can be alarger integer especially in ESI-MS.

9. Nominal Ion Mass: The mass of an ion containing the most abundant isotopes,calculated with nominal atomic weights.

10. Product Ions, Daughter Ions or Fragment Ions :All terms for ions formed byfragmentation of a precursor ion which may be the molecular ion.

11. Pseudo-Molecular Ions: Ions formed by FAB or chemical ionisation and othersoft ionisation methods, in which the molecular ion may be present with a proton

or other cations attached or, for negative ions, with a proton removed.

12. Radical Ion :An ion containing an unpaired electron.

13. u (Unified Atomic Mass Unit) :The symbol for the mass of a particle based on 12C= 12u exactly.

For better interpretation of the mass spectrum obtained from Mass Spectrometry, an individual musthave good knowledge about Chemical nature, Ionization processes & fragmentation patterns of the

compound. It is known that the detailed nature of the spectrum for a molecule depends upon its

Ionization potential, functionals groups it contains, the method of ionization, the sample pressure andtemperature and the instrument design.

Key Concepts of Data Interpretation

Some of the common approachs like Molecular ion, Fragmentation &Isotopic Abundance areexplained using some tyipcal examples for each.

Molecular Ion:

The molecular ion provides the molecular mass of the analyte and is the first clue used to interpret a

mass spectrum. The mass of the molecular ion is based up on the mass of the most abundant isotopefor each element in the molecule. This is not the atomic weight from the periodic table. Since many

mass spectrometers have unit mass resolution, the atomic mass is rounded to the nearest whole

number; this is called the nominal mass. For example the molecular ion for CHBr3 is observed atm/z 250, not at the formula weight of 253.


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Characteristics of Molecular Ions:

1) The m/z value must correspond to a reasonable molecular formula with the proper isotope

abundance.

2) Most compounds have an even molecular mass. The one common exception to this is the"Nitrogen Rule" discussed below.

3) The Nitro gen Rule: Any compound with an odd number of nitrogen atoms will have an oddmolecular mass. Any compound with an even number of nitrogen will have an even molecular mass.

This is because nitrogen is the only common atom where the most common isotope has an odd

valence and an even mass.

4) If a peak is t he molecular ion, the next highest mass fragment must correspond to the loss of a

possible neut ral fragment. For example, a peak that corresponds t o loss o f 5 u from the molecular ion i shighly unlikely.

In many mass spectra, the molecular ion is easily identified as the ion with the highest mass to chargeratio. However, this assignment should be made with caution because the highest mass to charge ion

be an impurity or an isotope of the molecular ion. In addition, many compounds fragment so easily

that no molecular ion is observed in the 70 eVEI spect rum. It is important to clarify t hat the molecular

ion is notnecessarily the ion with the greatest abundance, the ion with t he greatest abundance is the

base peak.

These are some of the characteristics of molecular ions to help you identify them in a mass spectrum.

Low energy EI or CI may be used to verify the molecular ion. As the ionization energy is reduced,

the molecular ion often increases in intensity. In chemical ionizat ion the adduct ion (M H+ M +1) is

observed at m/z.3

Example to be incorporated.

Fragmentation:

Although the molecular ion is useful for identification, it does not provide any structural information

about an unknown. The structural information is obtained from the fragmentation patterns of the

mass spectrum. Identifying an unknown without analyzing the fragmentation patterns is like putting

together a jigsaw puzzle without the picture. Fragmentation patterns are often complex, but they fittogether like pieces of the puzzle to identify the structure of the molecule.

Electron Ionization: ABCD + e- ABCD+ + 2 e-

Fragmentation:

Direct Cleavage ABCD+ AB+ + CD

Rearrangement ABCD+ AD+ + B=C

Fragmentation Mechanism: After a molecule is ionized, the molecular ion retains the excessionization energy. If this hexcess energy is greater than the energy required to break a chemical

bond, the molecule can fragment. The fragmentat ion processes are typically categorized as direct

cleavage or rearrangement (Figure 11.2). Cleavage reactions are simply the breaking of a bond to

produce two fragments. These reactions usually produce an even electron ion (AB+). The evenelectron ion is detected at an odd m/zvalue (assuming no nitrogen) and a neutral odd electron Thesereactions are thermodynamically favorable because they require less energy. However they also

3 CHBr3; (12 + 1 + 379) = 250. The mass of the molecular ion is based upon the isotope with the highest natural

abundance. The most common bromine isotope is 79Br. Do not use the weighted average atomic weight for Br (79.9)

which is based upon the natural abundance of different isotopes. The mass spectrum of CHBr also includes ions for other

naturally occurring isotopes. This includes m/z 252 (one 81Br, 49% natural abundance, and two 79Br), m/z 251 (one 13C,

1.1% natural abundance), m/z 257 (13C and three 81Br at the same time) and various other isotope combinations. The

intensity of each peak depends upon the probability for that combination of isotopes.


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require a concerted mechanism that is not as kinetically favorable when compared to a simple

cleavage reaction. Rearrangement ions easily identified because they are observed as odd electron

ions with an even m/zvalue. These fragments often provide important clues about the location andidentity of functional group s. The mass spectra of five different C10H22 isomers (Figure 11.2) show

how cleavage patterns help to identify a compound. The spectrum of n-decane includes the molecular

ion C10H22+ (m/z 142) and an evenly sp aced series of fragments. These fragments, with m/z 14separation, are formed by cleavage of the linear alky l chain at different locations. You should notice

the distribution of the fragments, C3 and C4 are the most abundant and very few long alky l chains

remain intact. This distribution depends up on the thermodynamics and kinetics of the fragmentation pathways. The four different methyl-nonane isomers have the same molecular ion as n-decane.

However, they have different fragmentation patterns because the position of the methyl group

changes the distribution of the fragmentation products. 2-M ethyl-nonane has three terminals CH 3 -groups so the loss of CH3 to produce C9H19

+(m/z127)

4is more likely than for other C10H22 isomers.

Loss of C2H5 to form C8H17+

(m/z113), however, is less likely for 2-methyl nonane since this loss is

only possible at one end of the molecule. As a result, the peak at m/z113 in the mass spectrum of 2-methyl nonane is only 0.5 % the intensity of the base peak

5(m/z43). A similar pattern is observed

for the loss of C3H7 in the mass spectrum of 3-methyl 3 nonane. The resulting fragment, C7H15+

(m/z

99), has a relative abundance of only 0.4 %. In 4- methyl nonane loss of C4H9 to produce C6H13+

(m/z85) has an abundance of only 7.1%. Loss of C5H11 to produce C5H11

+(m/z71) for 5-methyl nonane

has an abundance of 8.6%.

These five spectra demonstrate the importance of interpreting spectra. A computer search may not

distinguish these spectral features, but someone familiar with interpretation will realize thesignificance of the small missing peaks.

4Loss of CH3 (15u) from the molecular ion C10H22+ (m/z142), produces C9H19+ (m/z127)

5The base peak is the peak with the greatest abundance. The mass spectrum is usually normalized so that this

peak has an intensity of 100.


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Figure11.2 Fragmentation - 70 eV EI of C10H22 isomers. A) n-Decane, B) 2-methyl-nonane, C) 3-

methyl-nonane, D) 4-methyl-nonane, E) 5-methyl-nonane. (NOTE: mass spectra are shown with the

m/z along the x-axis and the abundance of each ion shown along the y-axis. The y-axis isnormalized to the base peak. This is the largest peak in the spectrum.

CH3

RR

NH+

H

H

CH3

RR

NH+

H

H

CH3

R

N+

H

H

+ R

Figure 11.3 Cleavage fragmentation of amines.

Functional groups can have a significant effect the fragmentation patterns observed in mass

spect rometry. For example, aliphatic amines p refer to undergo cleavage at the C-C bond to produce a

relatively stable CH2NH

2ion (Figure 11.3). The result ing fragments dist inguish primary,secondary,

and tertiary amines.

The mass spectra of three different n-pentaneamine isomers are shown in Figure 11.4- Pentaneamine

has an odd number of nitrogen atoms so the molecular ion (m/z87) has an odd mass to charge ratioand the cleavage fragment (m/z 30) has an even mass to charge ratio. Cleavage of 1-p entaneamine

produces CH2NH2 + (m/z30) and C4H9. The C4H9 fragment is not observed in the mass spectrum

because since this is a neut ral fragment.2-Pentaneamine has two cleavage sites. Loss of CH3 producesC4H8NH2 (m/z72) and loss of C3H7 produces C2H4NH2+ (m/z 44). Both of these these ions are observed

but the greater abundance of the m/z 44 signals indicated that loss of C3H7 is favored. The additional peak at

m/z 58 corresponds to C3H6NH2+, which could be formed by-cleavage loss of C2H5. 3-pentamine has two cleavage sites but they are symmetric so cleavage at either site results in loss of C2H5. Loss of C2H5 produces

C3H6NH2+(m/z 58), the basic peak in the mass spectrum.Now sp end some t imewith a piece of scratch

paper and interp ret some other peaks in these spectra.

Figure 11.4 70 eV EI spectra of pentaneamine isomers. A) 1-Pentaneamine, B) 2- Pentaneamine, C) 3-

Pentaneamine.


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Figure 11.5 Mclafferty rearrangement mechanism.

The McLafferty rearrangement (Figure 11.5), is a classic example of a rearrangement reaction. This

rearrangement results in formation of an intact neutral molecule and a radical ion with an even mass

to charge ratio. This reaction is significantly different from the cleavage reactions discussed

previously. The McLafferty rearrangement is often observed for carbony l compounds that contain a

linear alky l chain. If this alky l chain is long enough, a six-membered ring forms from the carbonyloxygen to the hydrogen on the fourth carbon. This spacing allows the hydrogen to transfer to the

carbonyl oxygen via a six membered ring. The McLafferty rearrangement is energetically favorable

because it results in loss of a neutral alkenes and formation of a resonance stabilized radical cation.

Figure 11.5 70 eV EI of C6H12O isomers. A) n-hexanal, B) 2- hexanone, C) 3- hexanone.

The products from the McLafferty rearrangement are observed in the mass spectra of C6 H12 O

isomers (Figure 11.5). The mass spectrum ofn-hexanal contains two even mass ions. C2 H4 O+ (m/z

44) is produced by the McLafferty rearrangement and C4 H8 + (m/z 56) is the McLaffertycompliment. The McLafferty compliment is produced when the charge is transferred to the alkenes

fragment during the rearrangement. The mass spectrum of 2-hexanone is easily distinguished from n-


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hexanal because the McLafferty rearrangement breaks a different C-C bond. This results in loss of

C3 H6 and produces C3 H 6O+ (m/z58). The mass spectrum of 3-hexanone does not have any major

even mass fragment ions so apparently the McLafferty rearrangement is not favorable.


Isotopic Abundance:

The existence of isotopes was first observed by Aston using a mass spectrometer to study neonions. When interpreting mass spectra it is important to remember that the atomic weight of an element

is a weighted average of the naturally occurring isotopes. M ass spectrometers separate these

isotopes and are even used to measure their relative abundance. Although this complicates the mass

spectrum, it also provides useful information for identifying the elements in an ion. Chlorine is anexcellent example of how isotope distributions are useful for interpretation. The molecular weight of

chlorine is 35.45u. This is calculated from the natural abundance of35Cl (75 %) and 37Cl(25%). The

natural abundance of these two isotopes is observed in the mass spectrum as two peaks separated bym/z 2 with a relative intensity of 3:1. The mass spectrum of CH3Cl (Figure 11.6) clearly shows two

peaks with the isotope distribution pattern for an ion with a single chlorine atom. CH3Cl+ (m/z 50)

and CH3Cl+ (m/z 52) are separated by m/z 2 and have the 3:1 abundance ratio characteristic of an

ion with a single chlorine atom.

Figure 11.6 Isotopic Abundance - 70 eV EI of CH3Cl.

If more than one chlorine atom is present, the isotope abundance is more complex. An ion with twochlorine atoms has three possible isotope combinations. This pattern is apparent in the mass

spectrum of CH2Cl2 (Figure). Ions are observed for CH235

Cl2+ (m/z 84), CH235

Cl37

Cl+ (m/z 86),and CH237Cl2+ (m/z 88). Based upon the probability of each combination of isotopes, the relative

intensity of these peaks is 10:6:1. The 3:1 isotope ratio for an ion with a single chlorine atom is

observed at m/z 49 and m/z 51. This corresponds to CH235

Cl+ and CH237

Cl+ fragments formed byloss of chlorine from the molecular ion. This spectrum also shows ions produced by loss of H+ and

H2.

Figure 11.7 Isotopic Abundance- 70 eV EI of CH2Cl2.

The 1.1% of natural abundance of 13C is another useful tool for interpreting mass spectra. The

abundance of a peak one m/z value higher, where a single 12C is replaced by a 13C, determined by


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the number of carbons in the ions. The rule of thumb for small compounds is that each carbon atom

in the ion increases the abundance of the M+1 peak by 1.1%. This effect is seen in all the spectra as

mentioned. In the n-decane mass spectrum (figure 11.7) compare the peak of12

C913

C1H22+ at m/z

143 (0.9% relative abundance) to the peak for 12C101H22 at m/z 142 (9% relative abundance). The

abundance of 13C peak is 10% the abundance of the 12C peak.

The relative abundance of isotopes of frequently encountered elements are given in the following

table.

Atom Isotope A Isotope A+1 Isotope A+2

Mass % Mass % Mass %

H 1 100 2 0.015

C 12 100 13 1.1

N 14 100 15 0.37

O 16 100 17 0.04 18 0.02

F 19 100

Si 28 100 29 5.1 30 3.4

P 31 100

S 32 100 33 0.80 34 4.4

Cl 35 100 37 32.5

Br 79 100 81 98.0

I 127 100

Table 11.1:Abundance of Isotopes for some common elements.


Summary

Data interpretation gives key information that can be used for identification of molecular structure:

information on the elemental composition, the molecular mass, the number of rings andinstaurations, the relationships between the structure and the fragmentations, etc. The user can use

this spectral data knowledge to identify unknown compounds, quantify unknown compound, and

identify structural and chemical properties of molecule.


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12. APPLICATIONS OF LCMS

Exploring with LCMS

Introduction

This topic provides an overview of various applications in LCMS. It covers some of the very

important applications of MS in the field of drug discovery & development, proteomics and

genomics, analysis of biomolecules, and pharmaceutical applications etc.

The application field of LC/MS is extremely large and is covered by a wide range of instruments and

techniques. Looking globally at the users, it is possible distinguish three groups, depending on howthey use LC/MS.

Users for which the main useful information from the mass spectrometer is themass information (molecular weight or fragments). The quantitative aspect is of no

or little importance.

Users for which the main interest is getting a very selective and sensitive detection.These users are targeting specific molecules. The quantitative aspect is important,

but the mass information is of secondary importance.

Users targeting specific molecules, wanting the quantification and the confirmationof the identity. The molecular weight and the presence of a few specific fragments,

which the expected abundance is as important as the sensitivity and selectivity

Various Applications of LCMS

Some of the important applications are mentioned below.

Analysis of Proteins:

Mass spectrometry is an important emerging method for the characterization of proteins. The two

primary methods for ionization of whole proteins are electrospray ionization and matrix-assisted

laser desorption ionization (MALDI). In keeping with the performance and mass range of availablemass spectrometers, two approaches are used for characterizing proteins. In the first, intact proteins

are ionized by either of the two techniques described above, and then introduced to a mass analyzer.

In the second, proteins are enzymatically digested into smaller peptides using an agent such astrypsin or pepsin. Other proteolytic digest agents are also used. The collections of peptide products

are then introduced to the mass analyzer. This is often referred to as the "bottom-up" approach of

protein analysis.


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Whole protein mass analysis is primarily conducted using either time-of-flight (TOF) MS, or Fourier

transform ion cyclotron resonance. These two types of instrument are preferable here because of their

wide mass range, and in the case of FT-ICR, its high mass accuracy. Mass analysis of proteolyticpeptides is a much more popular method of protein characterization, as cheaper instrument designs

can be used for characterization. Additionally, sample preparation is easier once whole proteins have

been digested into smaller peptide fragments. The most widely used instrument for peptide massanalysis is the quadrupole ion trap. Multiple stage quadrupole-time-of-flight and MALDI time-of-

flight instruments also find use in this application.

Analysis Of Nucleic Acids:

The use of mass spectrometry for the analysis of nucleic acids has come in two general areas, thefirst involving nucleosides, nucleotides and small oligonucleotides, and the second polynucleotides.

Only recently has ES and MALDI been applied with some success to polynucleotides analysis.

Applications include structure determination of modified bases from post-ribosomal processing ofRNA and DNA, and identification and quantitation of known bases in digests of nucleic acids using

mass spectrometry. The analysis of complex mixtures of nucleosides from RNA and DNA digests

has utilized LC/MS with thermospray, CF-FAB and electrospray ionization sources.

Pharmaceutical Applications:

Another important application area of LC-MS is the analysis of drugs and their metabolites. Possible

applications of LC-MS in the pharmaceuticals are given identifying by-products, degradationproducts, bioassay and metabolism. Pharmaceutical applications of LC-MS includes the analysis of

drugs for characterization and evaluation of LC-MS interfaces, the quantitation of drug metabolitesin the biological matrices, the identification of drug metabolites and drug conjugates and screening

of drugs and metabolites.

Isotope ratio MS:

Mass spectrometry is used to determine the isotopic composition of elements within a sample.

Differences in mass among isotopes of an element are very small, and the less abundant isotopes ofan element are typically very rare, so a very sensitive instrument is required. These instruments

sometimes referred to as isotope ratio mass spectrometers (IR-MS), usually use a single magnet to

bend a beam of ionized particles towards a series of Faraday cups, which convert particle impacts to

electric current. A fast on-line analysis of deuterium content of water can be done using Flowing

afterglow mass spectrometry, FA-MS. Probably the most sensitive and accurate mass spectrometerfor this purpose is the accelerator mass spectrometer (AMS). Isotope ratios are important markers ofa variety of processes. Some isotope ratios are used to determine the age of materials for example as

in carbon dating.

Pharmacokinetics:

Pharmacokinetics is often studied using mass spectrometry due to the complex nature of the matrix

(often blood or urine) and the need for high sensitivity to observe low dose and long time point data.The most common instrumentation used in this application is LC-MS with a triple quadrupole mass

spectrometer. LC/MS and LC/MS/MS have become the methods of choice for pharmacokinetic

studies, yielding concentration versus time data for drug compounds from in vivo samples such asplasma. Tandem mass spectrometry is usually employed for added specificity. Standard curves and

internal standards are used for quantitation of usually a single pharmaceutical in the samples. Thesamples represent different time points as a pharmaceutical is administered and then metabolized or

cleared from the body. Blank or t=0 samples taken before administration are important in

determining background and insuring data integrity with such complex sample matrices. Muchattention is paid to the linearity of the standard curve however it is not uncommon to use curve fitting

with more complex functions such as quadratics since the response of most mass spectrometers is

less than linear across large concentration range.


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Forensic Application:

LC/MS/MS is now widely used in confirmation analysis both in clinical and forensic toxicology. The

technique is particularly useful for polar, non-volatile, thermally labile compounds, which are

difficult to analyze by gas chromatography-based techniques. In addition, the sample pre-treatmentrequirements of LC/MS/MS make it an invaluable tool in high throughput toxicology laboratories. In

addition to confirmatory and quantitative analysis, LC/MS is also a powerful tool for drug screening.

The use of LC/MS/MS in clinical research laboratories is rapidly expanding and now includesapplications such as steroid analysis, homocysteine, methylmalonic acid and Vitamin D, amongothers

Metabolite ID:

The metabolism of a drug within a test animal can be extremely complex, involve multipleenzymatic pathways, and lead to a range of compounds with varying concentrations. Other drugs

have one or two major metabolic pathways that dominate their metabolism, but several minor

pathways can produce at least one metabolite. MS has emerged as an ideal technique for theidentification of such structurally diverse metabolites. When coupled with on-line HPLC, the

technique is extremely robust, rapid, sensitive and easily automated.

Common metabolic alterations can be predicted, and a list of expected metabolites can be compiled,

on the basis of a previously analyzed series. By combining this list with a list of suspectedmetabolites identified by precursor and neutral-loss scan data; a series of ions can be targeted for

product ion analysis. Any type of mass spectrometer capable of product ion scanning can be used atthis point, including a triple quadrupole.

Impurity Profiling:

A strong need for analytical methods to screen and quantify pesticide residues in agricultural

commodities, drinking and surfaces water and soil exists. As a result, there is a movement in theenvironmental community towards adopting LC/MS methods for the analysis of a much wider range

of analytes than presently required. LC/MS played an important role for the identification of

impurities contained in extracts and process intermediates. Because drugs derived from naturalsources often have a very diverse set of structural analogs, it is important to determine which analogs

are carried through the purification process and ultimately appear as impurities. This task presents a

unique challenge during the early stages of drug development due to the highly complex nature ofthe samples.

The LC/MS-based methods were significantly faster than the previously used analytical methods

based on scale-up, isolation, fractionation, and individual structural analysis. Software tools capableof sample tracking, interpretation, and data storage facilitate the structure profiling of impurities,

degradants, and metabolites.

Some other application of LCMS

Used for trace gas analysis of air, breath or liquid.

Used for Clinical studies: neonatal Screening, Hemoglobin analysis, drug testing.

The routine screening of common and prohibited drugs in blood/urine/saliva canalso be done. For e.g. detection of steroids in athletes.

Helped to determine the composition of molecular species found in space.

Used to locate oil deposits by measuring petroleum precursors in rocks.

LCMS is used for monitoring fermentation processes for biotechnology industries.

It is used to establish the elemental composition of semiconductor materials


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Used to determine gene damage from environmental causes.

Summary

Mass Spectrometry is powerful analytical tool that is used to identify unknown compounds, quantify

unknown compound, and identify structural and chemical properties of molecule. It also providesqualitative and quantitative determination of trace and ultra trace levels of organic compounds and

inorganic elements in different matrices and for understanding the physical and chemical properties

of different molecules. With this view, the technique provides valuable information in variousbranches of science viz. nuclear technology, chemistry, physics, biology, medicine, material science,

environment, forensic science, geochemistry, archeology, astronomy etc.


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13. SOFTWARE- ANALYST

Unassailable control of LCMS

Introduction

This topic provides overview of software related with mass spectrometer. It emphasis on 5 modes ofAnalyst software Configuration, Tuning, Acquisition, Explore and Quantitation. Each mode is

explained briefly with the help of a screen shot and some key point.

With such wide range of application, there was always a need felt for automating such technique.

The reasons of adapting automation in field of LCMS are:

High Throughput -large amount of data collected at same time.

Repeatability - repeating the user interaction with accuracy.

Reliability- reliable over results and confidence in the result

Consistency consistency in collecting accurate data.

To reduce human error and time

There are various software and software tools available in the market that can fulfill the above

criteria. Most software enables the user to control Mass Spectrometer and Peripheral devices such asHPLC system, Autosamplers, Pumps, Column Oven, valves. The easy to use interface allows the

user to perform all the activities necessary to analyze a sample. Such software can be used toconfigure the devices attached to instrument station. In addition it also performs some post

processing functions.

Analyst software is one such software, which helps the user to easily operate Mass Spectrometers

instruments. It performs all the major functions as mentioned above.


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Overview of the software

Analyst SW is divided into 5 sections, called as Modes that are separate functional areas-

1. Configure: It is used to set various options and parameters for the Analystapplication. In Configure mode, one can create, set or configure Security options,Hardware options, Reports etc.

2. Tune: Using Tune mode, one can set options for tuning the instrument to obtainoptimal results. In Tune mode, one can optimize resolution & quantitation, perform

mass calibration & manual tuning. Easy resolution optimization uses pre-defined

routines to automatically set peak width and mass calibration. Quantitativeoptimization automatically determines instrument settings for the best SIM signal

for accurate quantitative analysis of a compound. The powerful quantitative

capability of Analyst software allows rapid processing of quantitative data andautomatic extraction of critical information from data sets through queries and

metric plots.

3. Acquire: One can set options to determine how samples should be acquired. In Acquiremode, one can Build an acquisition method with Acquisition Method Editor or IDA wizard,Build a batch with the Batch Editor, monitor acquisition status etc.

4. Explore: One can perform qualitative analysis on samples. In Explore mode, one can viewgraph, chromatogram, spectrum, process data etc

5. Quantitate: Using Quantitate mode, one can perform activities related to quantitativeanalysis. For e.g. build quantitation method, review chromatographic peaks, review results

table etc.

Add-on tools available for different applications:

Library Search Tool Used to manage Library Database.

Fragmentation Interpretation Tool Used to create molecular structures.

Information Dependent Acquisition Wizard Allows to survey scan singleexperiment.


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Key Features

Following are the some example screen shots of key features of Analyst software:

Figure 13.1: Security


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Figure 13.2: Manual Tuning


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Figure 13.3a: Acquire Data Acquisition Method Editor


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Figure 13.3b: Acquire Data Acquisition Batch Editor


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Figure 13.4: Exploring data


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Figure 13.5a: Quantitative Create Quantitation Method


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Figure 13.5b: Quantitative Result Table


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Summary

To sum up,

Analyst software helps make your LC/MS tasks easier so that you can obtain the data you want in the

formats you need. The systems pump and autosampler are fully integrated and controlled as part ofthe method. Available LC devices are conveniently set up in the hardware configurator. Regardless

of the brand of LC module controlled, Analyst software provides the same easy to use control

interface.

Some key features available in Analysts software are:

Tune and calibration of instrument

Optimization of system for specific analytes or masses

Method builder.

Batch editors.

Exploring data

Quantitative analysis

Create and Print Reports

Audit Tracking


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14. References1. D. A. Skoog, Principles of Instrumental Analysis, third edition, Saunders

Publishing (1985)

2. H. M. McNair, L. N. Polite, HPLC, ACS Publication (1997)

3. L. R. Snyder, J. J. Kirkland, Introduction to modern liquid chromatography, second

edition, John Wiley and Sons, (1979)

4. L. R. Snyder, J. J. Kirkland, J. L. Glach, Practical HPLC method development,

second edition, John Wiley and Sons, (1997)

5. D.T. Rossi, M.W. Sinz ; Mass Spectrometry in Drug Discovery; Marcel dekker,Inc.

6. E.Hoffmann, V.Stroobannt ; Mass Spectrometry Principles and Applications, John

willey and Sons (2002)

7. M cLuckey , S.; Van Berkel, G.; Goeringer, D.; Glish, G.Anal. Chem. 1994, 66,

689A-695A.

8. M cLuckey , S.; Van Berkel, G.; Goeringer, D.; Glish, G.Anal. Chem. 1994, 66,

737A-743A.

9. Wilkins, C.L.; Gross, M .L.Anal. Chem. 1981, 53, 1661A-1676A.

10. Buchanan, M .V.; Hett ich, R.L.Anal. Chem. 1993, 65, 245A-259A.

11. M cLafferty , F.W.; Tureck, F.Interpretation of Mass Spectra; University ScienceBooks, 1993.

12. Davis, R. Mass Spectrometry/Analytical Chemistry by Open Learning; Wiley : New

York, 1987.


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15. Further Study

1. Warner, M .Anal. Chem. 1989, 61, 101A-103A.

2. DiFlip p o, F.; et. al.Phys Rev Lett. 1994, 73, 1482.

3. M unson, B.Anal. Chem. 1977, 49, 772A-778A.

4. M unson, B.; Field, F.J. Am. Chem. Soc., 1966, 88, 2621-2630.

5. Barber, M .; Bordoli, R.S.; Elliott , G.J .; Sedgwick, R.D.; Ty ler, A.N.Anal. Chem.

1982, 54, 645A-657A.

6. Fenselau, C.Anal. Chem. 1982, 54, 105A-114A.

7. Biemann, K.Anal. Chem. 1986, 58, 1288A-1300A.

8. Day , R.J.; Unger, S.e.; Cooks, R.G.Anal. Chem. 1980, 82, 557A-572A.

9. Winograd, N.Anal. Chem. 1993, 65, 622A-629A.

10. Benninghoven, A.; Hagenhoff, B.; Niehuis, E.Anal. Chem. 1993, 65, 630A-640A.

11. Huang, E.C.; Wachs, T.; Conboy , J .J.; Henion, J.D.Anal. Chem. 1990, 62, 713A-

725A.

12. Smith, R.D.; Wahl. J.H.; Goodlett, D.R.; Hofstadler, S.A.Anal. Chem. 1993, 65

574A-584A.

13. Hofstadler, S.; Bakhtiar, R.; Smith, R. J. Chem. Educ. 1996, 73, A82-A88.

14. Harrison, W.W.; Hess, K.R.; M arcus, R.K.; King, F.L.Anal. Chem. 1986, 58,

341A-356A.

15. Houk, R.S.Anal. Chem. 1986, 58, 97A-105A.

16. Vela, N.P.; Olson, L.K.; Caruso, J.A.Anal. Chem. 1993, 65, 585A-597A.

17. Karas, M .; Hillenkamp , F.Anal. Chem. 1988, 60, 2299-2301.

18. Fenselau, C.Anal. Chem. 1997, 69, 661A-665A.

19. Lattimer, R.P.; Schulten, H.R.Anal. Chem. 1989, 61, 1201A-1215A.

20. M acfarlane, R.D.Anal. Chem. 1983, 55, 1247A-1264A.

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LCMS Introduction Part 2

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