Basics of LC/MS

Primer

Contents

Why Liquid Chromatography/Mass Spectrometry? 4 Instrumentation 6 Ion Sources 6 Electrospray ionization 7 Atmospheric pressure chemical ionization 8 Atmospheric pressure photoionization 9 Mass Analyzers 10 Quadrupole 10 Time-of-flight 11 Ion trap 11 Fourier transform-ion cyclotron resonance (FT-ICR) 12 Collision-Induced Dissociation and Multiple-Stage MS 13 CID in single-stage MS 14 CID and multiple-stage MS 14 Adapting LC Methods 16 Sample preparation 17 Ionization chemistry 17 Capillary LC/MS and CE/MS 19 Applications 20 Molecular Weight Determination 20 Differentiation of similar octapeptides 20 Determining the molecular weight of green fluorescent protein 21 Structural Determination 22 Structural determination of ginsenosides using MSn analysis 22 Pharmaceutical Applications 24 Rapid chromatography of benzodiazepines 24 Detection of degradation products for salbutamol 24 Identification of bile acid metabolites 25 Biochemical Applications 26 Rapid protein identification using capillary LC/MS/MS and database searching 26 Clinical Applications 28 High-sensitivity detection of trimipramine and thioridazine 28 Food Applications 28 Identification of aflatoxins in food 28 3 Determination of vitamin D3 in poultry feed supplements using MS 30 Environmental Applications 32 Detection of phenylurea herbicides 32 Detection of low levels of carbaryl in food 32 CE/MS Applications 34 Analysis of peptides using CE/MS/MS 34 Why Liquid Chromatography/ Mass spectrometers also generate three- Mass Spectrometry? dimensional data. In addition to signal strength, they generate mass spectral Liquid chromatography is a fundamental data that can provide valuable information separation technique in the life sciences about the molecular weight, structure, and related fields of chemistry. Unlike gas identity, quantity, and purity of a sample. chromatography, which is unsuitable for Mass spectral data add specificity that nonvolatile and thermally fragile molecules, increases confidence in the results of both liquid chromatography can safely qualitative and quantitative analyses. separate a very wide range of organic compounds, from small-molecule drug R metabolites to peptides and proteins.

Traditional detectors for CH CH CH NHCOCH liquid chromatography 2 2 2 3 CH2CH2CH2OH include refractive index, CH2CH2CH(NH2)COOH 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. % Rel. Abundance% Rel. Abundance % Rel.

m/z

Figure 1. Two-dimensional abundance data and three-dimensional mass spectral data from a mass spectrometer

4 For most compounds, a mass spectrometer is Some mass spectrometers have the ability to more sensitive and far more specific than all perform multiple steps of mass spectrometry other LC detectors. It can analyze compounds on a single sample. They can generate a that lack a suitable chromophore. It can also mass spectrum, select a specific ion from identify components in unresolved chromato- that spectrum, fragment the ion, and generate graphic peaks, reducing the need for perfect another mass spectrum; repeating the entire chromatography. cycle many times. Such mass spectrometers can literally deconstruct a complex molecule Mass spectral data complements data from piece by piece until its structure is determined. 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.

1200000 800000 MS TIC 400000

4000 m/z 2000 648

12000 8000 m/z 4000 376

300 200 m/z 100 1325

5 10152025303540min.

Figure 2. Identification of three components in a chromatographically unresolved peak

5 Instrumentation molecules from the analyte molecules (direct liquid inlet, thermospray) or did so before ion- Mass spectrometers work by ionizing mole- ization (particle beam). The analyte molecules cules and then sorting and identifying the ions were then ionized in the mass spectrometer according to their mass-to-charge (m/z) ratios. under vacuum, often by traditional electron Two key components in this process are the ionization. These approaches were successful ion source, which generates the ions, and the only for a very limited number of compounds. mass analyzer, which sorts the ions. Several different types of ion sources are commonly The introduction of atmospheric pressure used for LC/MS. Each is suitable for different ionization (API) techniques greatly expanded classes of compounds. Several different types the number of compounds that can be suc- of mass analyzers are also used. Each has cessfully analyzed by LC/MS. In atmospheric advantages and disadvantages depending on pressure ionization, the analyte molecules the type of information needed. are ionized first, at atmospheric pressure. The analyte ions are then mechanically Ion Sources and electrostatically separated from neutral molecules. Common atmospheric pressure Much of the advancement in LC/MS over the ionization techniques are: last ten years has been in the development of ion sources and techniques that ionize the • Electrospray ionization (ESI) analyte molecules and separate the resulting • Atmospheric pressure chemical ionization ions from the mobile phase. (APCI) • Atmospheric pressure photoionization (APPI) Earlier LC/MS systems used interfaces that either did not separate the mobile phase

Figure 3. Applications of various 100,000 LC/MS ionization techniques Electrospray Ionization

10,000

1000 APPI APCI Molecular Weight 100

10 Nonpolar Very Polar Polarity

6 Electrospray ionization Some gas-phase reactions, mostly proton transfer and charge exchange, can also Electrospray relies in part on chemistry to gen- occur between the time ions are ejected erate analyte ions in solution before the analyte from the droplets and the time they reach reaches the mass spectrometer. The LC eluent the mass analyzer. is sprayed (nebulized) into a chamber at atmospheric pressure in the presence of a strong Electrospray is especially useful for analyzing electrostatic field and heated drying gas. large biomolecules such as proteins, peptides, and oligonucleotides, Ions but can also analyze Nebulizer gas smaller molecules like benzodiazepines and sulfated Heated nitrogen drying gas conjugates. Solvent spray Large molecules + + + often acquire more + + + + + + + + + + + + + + than one charge. Thanks to this multiple charging, Dielectric capillary entrance electrospray can be used to analyze molecules as large Figure 4. Electrospray ion source as 150,000 u even though the mass range (or more accurately The electrostatic field causes further mass-to-charge range) for a typical LC/MS dissociation of the analyte molecules. instruments is around 3000 m/z. For example: The heated drying gas causes the solvent 100,000 u / 10 z = 1,000 m/z in the droplets to evaporate. As the droplets shrink, the charge concentration in the When a large molecule acquires many charges, droplets increases. Eventually, the repulsive a mathematical process called deconvolution force between ions with like charges exceeds is often used to determine the actual molecular the cohesive forces and ions are ejected weight of the analyte. (desorbed) into the gas phase. These ions are attracted to and pass through a capillary sampling orifice into the mass analyzer.

Evaporation Analyte ion ejected Figure 5. Desorption of ions from solution + + + + - +++ + + + ++- ++ - - + - -+ + - -- + + - -+ +- -++ - - - + +--+ ++ + ++ + ++ + -- + + ++-+

7 Atmospheric pressure APCI is applicable to a wide range of polar chemical ionization and nonpolar molecules. It rarely results in multiple charging so it is typically used for In APCI, the LC eluent is sprayed through molecules less than 1,500 u. Due to this, a heated (typically 250°C – 400°C) vaporizer and because it involves high temperatures, at atmospheric pressure. The heat vaporizes APCI is less well-suited than electrospray the liquid. The resulting gas-phase solvent for analysis of large biomolecules that may molecules are ionized by electrons discharged be thermally unstable. APCI is used with from a corona needle. The solvent ions then normal-phase chromatography more often transfer charge to the analyte molecules than electrospray is because the analytes are through chemical reactions (chemical ioniza- usually nonpolar. tion). The analyte ions pass through a capillary sampling orifice into the mass analyzer.

Figure 6. APCI ion source HPLC inlet

Nebulizer (sprayer) Nebulizing gas

Vaporizer (heater)

Drying gas

+ + + + + + + + + + + + + + +

Corona discharge needle Capillary

8 Atmospheric pressure photoionization APPI is applicable to many of the same compounds that are typically analyzed by Atmospheric pressure photoionization (APPI) APCI. It shows particular promise in two for LC/MS is a relatively new technique. As applications, highly nonpolar compounds in APCI, a vaporizer converts the LC eluent to and low flow rates (<100 µl/min), where APCI the gas phase. A discharge lamp generates sensitivity is sometimes reduced. photons in a narrow range of ionization energies. The range of energies is carefully In all cases, the nature of the analyte(s) chosen to ionize as many analyte molecules and the separation conditions have a strong as possible while minimizing the ionization influence on which ionization technique: of solvent molecules. The resulting ions pass electrospray, APCI, or APPI, will generate through a capillary sampling orifice into the the best results. The most effective technique mass analyzer. is not always easy to predict.

Figure 7. APPI ion source HPLC inlet

Nebulizer (sprayer) Nebulizing gas

Vaporizer (heater)

UV lamp Drying gas

hv + + + + + + + + + + + + + + +

Capillary

9 Mass Analyzers through the filter at a given time. Quadrupoles tend to be the simplest and least expensive Although in theory any type of mass analyzer mass analyzers. could be used for LC/MS, four types: • Quadrupole Quadrupole mass analyzers can operate in • Time-of-flight two modes: • Ion trap • Scanning (scan) mode • Fourier transform-ion cyclotron resonance • Selected ion monitoring (SIM) mode (FT-ICR or FT-MS) In scan mode, the mass analyzer monitors a are used most often. Each has advantages and range of mass-to-charge ratios. In SIM mode, disadvantages depending on the requirements the mass analyzer monitors only a few mass- of a particular analysis. to-charge ratios.

Quadrupole SIM mode is significantly more sensitive than scan mode but provides information about A quadrupole mass analyzer consists of four fewer ions. Scan mode is typically used for parallel rods arranged in a square. The analyte qualitative analyses or for quantitation when ions are directed down the center of the all analyte masses are not known in advance. square. Voltages applied to the rods generate SIM mode is used for quantitation and monitor- electromagnetic fields. These fields determine ing of target compounds. which mass-to-charge ratio of ions can pass

Figure 8. Quadrupole mass analyzer To detector

From ion source 1 Scan Mass Range Scan

m/z Abundance

time m/z

SIM 1 Scan Discrete Masses

m/z Figure 9. The quadrupole mass

analyzer can scan over a range Abundance of mass-to-charge ratios or time m/z alternate between just a few

10 Time-of-flight travel faster and arrive at the detector first, so the mass-to-charge ratios of the ions are In a time-of-flight (TOF) mass analyzer, a determined by their arrival times. Time-of- uniform electromagnetic force is applied to flight mass analyzers have a wide mass all ions at the same time, causing them to range and can be very accurate in their accelerate down a flight tube. Lighter ions mass measurements.

Ion trap Flight tube (field-free region) An ion trap mass analyzer consists of + a circular ring electrode plus two end Repeller + Detector caps that together form a chamber. Ions entering the chamber are “trapped” there by electromagnetic fields. Another field can be applied to selectively eject ions Accumulation from the trap.

From ion source Ion traps have the advantage of being able Flight tube (field-free region) to perform multiple stages of mass spectrometry without additional mass analyzers.

+ Repeller Detector

Ejection

+ + + + + + + +

Abundance Accumulation Ejection

m/z

Figure 10. Time-of-flight mass analyzer Abundance

m/z

Figure 11. Ion trap mass analyzer

11 Fourier transform-ion Like ion traps, FT-ICR mass analyzers can cyclotron resonance (FT-ICR) perform multiple stages of mass spectrometry An FT-ICR mass analyzer (also called FT-MS) without additional mass analyzers. They also is another type of trapping analyzer. Ions have a wide mass range and excellent mass entering a chamber are trapped in circular resolution. They are, however, the most expen- orbits by powerful electrical and magnetic sive of the mass analyzers. fields. When excited by a radio-frequency (RF) electrical field, the ions generate a time- dependent current. This current is converted by Fourier transform into orbital frequencies of the ions which correspond to their mass-to- charge ratios.

Figure 12. FT-ICR mass analyzer +

12 Collision-Induced Dissociation and Multiple-Stage MS 279.1

CH3 + The atmospheric pressure ionization 350000 [M + H] techniques discussed are all O N 300000 O S relatively “soft” techniques. They NH N CH3 generate primarily: 250000 • Molecular ions M+ or M– H N 200000 2 • Protonated molecules [M + H]+ • Simple adduct ions [M + Na]+ 150000

• Ions representing simple losses 100000

such as the loss of a water 280.0 + + [M + Na] [M + H – H2O] 50000 301.0 281.0

0 The resulting molecular weight 100 200 300 m/z information is very valuable, but complementary structural information is often needed. To obtain Figure 13. Mass spectrum of structural information, analyte ions are sulfamethazine acquired without collision-induced fragmented by colliding them with neutral dissociation exhibits molecules in a process known as collision- little fragmentation induced dissociation (CID) or collisionally activated dissociation (CAD). Voltages are applied to the analyte ions to add energy to the collisions and create more fragmentation.

Figure 14. Mass spectrum m/z 156 of sulfamethazine acquired

124.1 CH3 m/z 186 with collision-induced O N dissociation exhibits more O S fragmentation and thus 80000 NH N CH3 more structural information 186.0 m/z 108

279.1 m/z 213 + 60000 [M + H] H2N

156.1 m/z 124

40000

108.2 + [M + Na] 301.0

20000 323.0 213.2 107.1 280.1 125.1 187.0 157.1

0 100 200 300 m/z

13 CID in single-stage MS product ions came from which precursor ion. The resulting spectra may include mass peaks CID is most often associated with multistage from background ions or coeluting compounds mass spectrometers where it takes place as well as those from the analyte of interest. between each stage of MS filtering, but CID This tradeoff may be acceptable when analyz- can also be accomplished in single-stage ing relatively pure samples, but does not give quadrupole or time-of-flight mass spectrome- good results if chromatographic peaks are not ters. In single-stage mass spectrometers, well resolved or background levels are high. CID takes place in the ion source and is thus sometimes called source CID or in-source CID. CID and multiple-stage MS Analyte (precursor) ions are accelerated and collide with residual neutral molecules to yield Multiple-stage MS (also called tandem MS or fragments called product ions. MS/MS or MSn) is a powerful way to obtain structural information. In triple-quadrupole or The advantage of performing CID in single- quadrupole/quadrupole/time-of-flight instru- stage instruments is their simplicity and ments (see Figure 16), the first quadrupole is relatively low cost. The disadvantage is that used to select the precursor ion. CID takes ALL ions present are fragmented. There is place in the second stage (quadrupole or no way to select a specific precursor ion octopole), which is called the collision cell. so there is no sure way to determine which

Figure 15. In-source LC/MS CID with a single- quadrupole mass analyzer Abundance

Capillary CID Q1 m/z

LC/MS/MS Abundance CapillaryQ1 Q2 (CID) Q3 m/z

Figure 16. MS/MS in a triple-quadrupole mass spectrometer

14 The third stage (quadrupole or TOF) then A major advantage of multiple-stage MS generates a spectrum of the resulting is its ability to use the first stage of MS to product ions. It can also perform selected discard nonanalyte ions. Sample cleanup ion monitoring of only a few product ions and chromatographic separation become when quantitating target compounds. much less critical. With relatively pure samples, it is quite common to do away In ion trap and FT-ICR mass spectrometers, with chromatographic separation altogether all ions except the desired precursor ion and infuse samples directly into the mass are ejected from the trap. The precursor spectrometer to obtain product mass spectra ion is then energized and collided to generate for characterization or confirmation. product ions. The product ions can be ejected to generate a mass spectrum, or a particular product A. B. ion can be retained and collided to obtain another set of product ions. This process can be sequentially + automated so that the most + + + abundant ion(s) from each stage of MS are retained and collided. This is a very powerful technique for determining the structure of Accumulation Nonprecursor ions ejected molecules. It is also a powerful technique for obtaining peptide C. D. mass information that relates to the sequence of amino acids in a peptide. + + + + + + +

Collision Product ions ejected and detected Abundance

Figure 17. MS/MS in an ion m/z trap mass spectrometer

15 Adapting LC Methods Changes to LC methods required for modern LC/MS systems generally involve changes in Early LC/MS systems were limited by funda- sample preparation and solution chemistry to: mental issues like the amount of LC eluent the mass spectrometer could accept. Significant • Ensure adequate analyte concentration changes to LC methods were often required to • Maximize ionization through careful adapt them to MS detectors. selection of solvents and buffers • Minimize the presence of compounds that Modern LC/MS systems are more versatile. compete for ionization or suppress signal Many mass spectrometers can accept flow through gas-phase reactions rates of up to 2 ml/min. With minor modifica- tions, the same instruments can also generate good results at microliter and nanoliter flow rates. Ion sources with orthogonal (off-axis) nebulizers are more tolerant of nonvolatile buffers and require little or no adjustment, even with differing solvent compositions and flow rates.

Figure 18. Salt deposits in this Agilent APCI ion source had little effect on performance thanks to orthogonal spray orientation and robust ion source design

16 Sample preparation • Use solvents that have low heats of vapor- ization and low surface tensions to enhance Sample preparation generally consists of ion desorption concentrating the analyte and removing compounds that can cause background ions • Make sure that gas-phase reactions do or suppress ionization. Examples of sample not neutralize ions through proton transfer preparation include: or ion pair reactions • On-column concentration to increase If pH adjustments interfere with proper analyte concentration chromatography, postcolumn modification • Desalting to reduce the sodium and of the solvent may be a good solution. This potassium adduct formation that can improve MS response without compro- commonly occurs in electrospray mising chromatography. • Filtration to separate a low- molecular-weight drug from proteins in plasma, milk, or tissue +13 pH 2.5 Ionization chemistry +12 +14 Because formation of analyte ions in solution is essential to achieving +11 good electrospray results, careful +15 +10 attention must be paid to proper +9 solution chemistry. For electrospray: • Select more volatile buffers to +12 +11 reduce the buildup of salts in the ion source +10 pH 6 • Adjust solvent pH according to the +13 polarity of ions desired and the pH +9 of the sample +14 +8 +15

pH 12

Figure 19. Effect of solvent pH on the abundance of multiply charged ions of the protein Lysozyme in electrospray mode

17 Solution chemistry is less critical for APCI Vaporizer temperature also affects APCI operation because ionization occurs in the ionization results. The temperature must be gas phase, not the liquid phase, but solvent hot enough to vaporize the solvent but not selection can still have a significant effect on so hot as to cause thermal degradation of the APCI analyte signal response. analyte molecules. • Select more volatile solvents • Select solvents with a lower charge affinity than the analyte • Protic solvents generally work better than nonprotic solvents for positive ion mode • For negative ionization, solvents that readily capture an electron must be used • Ammonium salts in the mobile phase can cause ammonium adduct formation

200˚C

1 2 3 4 56 7 8910 11 12 13 14 15 16

400˚C

1 2 3 4 567 89 1011 12 13 14 15 16

Figure 20. APCI analysis with an inadequate vaporizer temperature (200°C) yields poor results for some compounds compared to a more typical vaporizer temperature (400°C)

18 Capillary LC/MS and CE/MS Capillary electrophoresis (CE) is a technique with a high separation efficiency and the Capillary LC and capillary electrophoresis added benefit of being able to handle very (CE) are commonly used alongside HPLC. complex matrices. It has proven useful for Capillary LC at microliter to nanoliter flow such diverse samples as: peptides, drugs of rates often provides better sensitivity than abuse, drugs in natural products, flavanoids, conventional flow rates for extremely small and aromatic amines. sample quantities. It is commonly used for protein and peptide analysis but has also With specialized nebulizers and method proven very useful for the analysis of small adjustments, electrospray ionization can be quantities of drugs. used for both capillary LC/MS and CE/MS. The mass spectrometry benefits of selectivity and sensitivity are available to both of these separation techniques.

2.0 Base Peak 50 mM NaPO3 Salbutamol Terbutaline 5 µg/mL 1.5 5 µg/mL HO HO OH 1.0 NH HO NH OH HO % Relative Abundance 0.5

0.0 2 4 6 8 10 12 Time (min)

Figure 21. CE/MS/MS analysis of terbutaline and salbutamol shows good chromatographic separation and good signal even in the presence of a high concentration of sodium phosphate salt

19 Applications Differentiation of similar octapeptides LC/MS is suitable for many applications, from Figure 22 shows the spectra of two peptides pharmaceutical development to environmental whose mass-to-charge ratios differ by only analysis. Its ability to detect a wide range 1 m/z. The only difference in the sequence of compounds with great sensitivity and speci- is at the C-terminus where one peptide has ficity has made it popular in a variety of fields. threonine and the other has threonine amide. The smaller fragments are identical in the two Molecular Weight Determination spectra, indicating that large portions of the One fundamental application of LC/MS is two peptides are very similar. The larger frag- the determination of molecular weights. This ments contain the differentiating peptides. information is key to determining identity.

Figure 22. Mass spectra differentiating two very similar octapeptides 100 Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr 858.5 80

40 739.3 880.3

20 444.2 576.3 498.2 841.3 343.1 397.2 0 857.5 100 Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr amide 80

40 879.5 739.3 840.3

20 576.3 444.2 379.1 0

400 600 800 m/z

20 Determining the molecular weight The upper part of the display in Figure 23 of green fluorescent protein shows the full scan mass spectrum of GFP. The pattern of mass spectral peaks is charac- Green fluorescent protein (GFP) is a 27,000- teristic of a multiply charged analyte. Each Dalton protein with 238 amino acids. It emits peak represents the molecule with a different a green light when excited by ultraviolet light. number of charges. The lower display is a deconvoluted mass spectrum generated by During electrospray ionization, GFP acquires the data system for the singly charged analyte. multiple charges. This allows it to be analyzed by a mass spectrometer with a relatively limited mass (mass-to-charge) range. Mass deconvolution is then used to determine the molecular weight of the protein.

Figure 23. Molecular weight determination of green fluorescent 789.95

protein by electro- 839.45 spray LC/MS 120000 866.45 100000 767.55 895.45 746.15 80000 926.15 959.15 60000 726.25 994.85 1032.95 40000 1074.15 1118.85 1167.55 1220.45 1278.75

20000 1342.65 1413.25 1579.65

1000 1500

1000000

800000 Deconvoluted spectrum 600000 MW = 26828.84

400000

200000

26700 26800 26900 27000

21 Structural Determination MSn analysis in an ion trap mass spectrometer permits multiple stages of precursor ion Another fundamental application of LC/MS isolation and fragmentation. This stepwise is the determination of information about fragmentation permits individual fragmentation molecular structure. This can be in addition pathways to be followed and provides a great to molecular weight information or instead deal of structural information. of molecular weight information if the identity of the analyte is already known. Figure 24 shows the full scan mass spectrum from a direct infusion of the ginsenoside Rb1. Structural determination of The most prominent feature is the sodium ginsenosides using MSn analysis adduct ion [M + Na]+ at m/z 1131.7. MS/MS Ginseng root, a traditional Chinese herbal of m/z 1131.7 yields a product ion at m/z 789.7 remedy, contains more than a dozen corresponding to cleavage of a single glyco- biologically active saponins called ginseno- sidic bond (Figure 25). Subsequent isolation sides. Since most ginsenosides contain and fragmentation of m/z 789.7 (Figure 26) multiple oligosaccharide chains at different yields two products: a more abundant ion at positions in the molecule, structural elucidation m/z 365.1 corresponding to loss of the oligo- of these compounds can be quite complicated. saccharide chain (–Glc 2 –Glc), and a less abundant ion at m/z 627.5 representing the loss of a deoxyhexose sugar.

Figure 24. Full scan mass spectrum of ginsenoside Rb1 showing primarily 100 HO 1131.7 O O sodium adduct ions OH O OH O OH OH OH 80 OH OH [M + Na]+ m/z 1131.7

60 HO O O OH HO HO 40 O O OH % Relative Abundance HO OH 20

0 200 400 600 800 1000 1200 m/z

22 Figure 25. Full scan product ion (MS/MS) HO 789.7 spectrum from the sodium 100 O O MS/MS of 1131.7 OH O adduct at m/z 1131.7 OH O OH OH OH OH m/z 789.7 80 OH [M + Na]+ m/z 1131.7 60 HO O O OH HO HO O m/z 365.2 O 40 OH

% Relative Abundance OH

20 365.2

0 1131.7

200 400 600 800 1000 1200 m/z

Figure 26. Subsequent full scan product ion spectrum (MS3) from 365.1 1131.7 789.7 the ion at m/z 789.7 100 OH Na+ m/z 627.5 80 m/z 365.1

HO O O 60 OH

HO OH O O OH

40 OH

% Relative Abundance OH

20 627.5

0 200 300 400 500 600 700 800 m/z

23 Pharmaceutical Applications Identification of bile acid metabolites Rapid chromatography of benzodiazepines The MSn capabilities of the ion trap mass spectrometer make it a powerful tool for The information available in a mass spectrum the structural analysis of complex mixtures. allows some compounds to be separated Intelligent, data-dependent acquisition tech- even though they are chromatographically niques can increase ion trap effectiveness unresolved. In this example, a series of benzo- and productivity. They permit the identification diazepines was analyzed using both UV and of minor metabolites at very low abundances MS detectors. The UV trace could not be from a single analysis. One application is used for quantitation, but the extracted ion the identification of metabolic products of chromatograms from the MS could be used. drug candidates. The mass spectral information provides additional confirmation of identity. Chlorine has a characteristic pattern because of the relative abundance of the two most abundant isotopes. In Figure 27, the triazolam spectrum shows that triazolam has two chlorines and the diazepam spectrum shows that diazepam has only one.

Figure 27. MS identification Rapid chromatography and isotopic information and quantification of individual benzodiazepines mAU from an incompletely 2 UV resolved mixture 1.5

1 343.1 0.5 Triazolam 0 345.1 2 Cl 1234min

350 MS 300 Triazolam 250 285.2 Clobazam 200 Diazepam Diazepam

150 Alprazolam 287.2 1 Cl 100 Estrazolam Lorazepam 50 250 300 350 oxazepam Diazepam 0 1234min

24 This example uses the in vitro incubation of corresponding to a predicted minor metabolite the bile acid deoxycholic acid with rat liver (cholic acid) that eluted at 9.41 minutes. The microsomes to simulate metabolism of a full scan MS/MS product spectrum (Figure 28C) drug candidate. Intelligent, data-dependent from the ion at m/z 407 confirms the identity. acquisition was used to select the two most abundant, relevant ions in each MS scan. Significant time was saved because the These precursor ions were automatically confirming MS/MS product ion spectra were fragmented and full scan product ion acquired automatically in the same run as the spectra collected. full scan MS data.

Figure 28A shows the base peak chromatogram. Figure 28B shows the extracted ion chromatogram of the [M-H]– ion at m/z 407

Figure 28. Identification of a minor metabolite of deoxycholic acid 11.64 A through MS/MS 5 Base Peak: m/z 150–500 4 12.01 Minor Metabolite 3 12.28 10.24 2 0.77 13.49

0 2 4 6 8 10 12 14

4 9.41 B

3 m/z 407

0 24681012 14

289.5 100 343.4 C H O OH MS/MS of 407 80 OH H at 9.41 min 60 H 325.3 389.5 40 HH OH OH H 20 Cholic Acid 0 100 200 300 400 500 m/z

25 Biochemical Applications In this example, a capillary LC and ion trap mass spectrometer were used to acquire Rapid protein identification data from a mixture of five tryptically digested using capillary LC/MS/MS proteins at a concentration of 1 pmol/µl each and database searching (Figure 29). Using intelligent, automated data- Traditional methods of protein identification dependent acquisition, a full scan product generally require the isolation of individual ion (MS/MS) spectrum was acquire from the proteins by two-dimensional gel electro- most abundant relevant ion in each mass scan phoresis. The combination of capillary throughout the entire run. All MS and MS/MS LC/MS/MS with intelligent, data-dependent data were acquire from a single analysis. acquisition and probability-based database searching makes it possible to rapidly identify as many as 100 proteins in a single analysis.

Intens. 18.39 x107 17.55

0.8 31.37 0.6 13.43 30.37 Base Peak: 14.43 0.4 4.43 20.59 m/z 150–2000 11.75 21.09 0.2 22.35

0.0 5 10 15 20 25 30 35 Time [min]

Figure 29. Base peak chromatogram generated from 1 pmol total material injected on column

26 Protein identification was accomplished using MASCOT software that correlated the uninterpreted MS/MS data with sequences in a database. Figure 30 demonstrates the excellent match between the observed MS/MS spectrum from the most abundant ion (m/z 807.2) in the chromatographic peak at 17.55 minutes and the theoretical y-ion series predicted for a tryptic peptide from LLDNWDSVTSTFSK human apolipoprotein, one of the proteins in Theoretical 971.5 the sample mixture. 670.3 856.4 Y(9) 1386.6 381.2 569.3 Y(8) Y(6) Y(12) Y(5) Y(3) 1157.5 769.4 1271.6 Y(10) Y(7) Y(11)

971.6 100 400 600 800 1000 1200 1400 670.4

80 856.5

1386.7 60 Observed 381.3 569.3 40 769.5 MS/MS of m/z 807.2 % Relative Abundance 1271.5 20 1157.5

0 200 600 1000 1400 1800 m/z

Figure 30. Full scan MS/MS spectra from the doubly charged parent ion m/z 807.2 and the matching theoretical sequence identified by database searching

27 Clinical Applications Food Applications High-sensitivity detection of Identification of aflatoxins in food trimipramine and thioridazine Aflatoxins are toxic metabolites produced in For most compounds, MS is more sensitive foods by certain fungi. Figure 32 shows the than other LC detectors. Trimipramine is total ion chromatogram from a mixture of four a tricyclic antidepressant with sedative aflatoxins. Even though they are structurally properties. Thioridazine is a tranquilizer. very similar, each aflatoxin can be uniquely Figure 31 shows these compounds in a urine identified by its mass spectrum (Figure 33). extract at a level that could not be detected by UV. To get the maximum sensitivity from a single-quadrupole mass spectrometer, the analysis was done by selected ion monitoring.

Figure 31. Trimipramine and thioridazine in a urine extract mAU UV 200 280 nm 0

80000 Trimipramine EIC m/z 295 0

40000 Trimipramine EIC m/z 100 0

20000 EIC Thioridazine m/z 371 0 8000 EIC Thioridazine m/z 126 0 123456min

28 Figure 32. Total ion chromatogram of a Scan of 2 ng mixture of aflatoxins 4 TIC 200000

160000 1) Aflatoxin G2 3 2 1 120000 2) Aflatoxin G1

3) Aflatoxin B2 80000 4) Aflatoxin B1

40000

6.00 8.00 10.00 12.00 14.00 16.00 18.00

Figure 33. Unique mass spectra 315 allow positive + 331 [M+H] [M+H]+ identification 90 Aflatoxin G2 90 Aflatoxin B2 of structurally OO 313 O O + similar aflatoxins O O [M-OH] O 50 50 353 O O + 337 OCH3 O O [M+Na] OCH3 287 + [M+Na]

120 160 200 240 280 320 360 120 160 200 240 280 320

329 313 + [M+H] + [M+H] Aflatoxin B1 90 Aflatoxin G1 311 90 O O [M-OH]+ O O OO 50 50 O 351 285 335 O O 243 + + OCH3 [M+Na] O 283 O OCH3 [M+Na]

120 160 200 240 280 320 360 120 160 200 240 280 320

29 Determination of vitamin D3 in preparation and derivatization. Further, the poultry feed supplements using MS3 multiple-stage MS capability of the ion trap eliminates the need for chromatographic Vitamin D is an essential constituent in human separation, greatly speeding analyses. and animal nutrition. Livestock diets deficient in vitamin D can cause growth abnormalities. Flow injection analysis of a poultry feed extract yields a peak at m/z 385 suggesting Traditional GC/MS analysis methods for the presence of vitamin D . Isolation and vitamin D in feed extracts require extensive 3 3 fragmentation of the precursor ion at m/z 385 and time-consuming sample preparation and is inconclusive. The full scan product ion derivatization prior to analysis. Atmospheric spectrum shows a prominent peak at m/z 367 pressure chemical ionization with ion trap representing the loss of a single water mole- detection provides a sensitive analytical cule but little other fragmentation (Figure 34). method without the need for extensive sample

m/z 367 367.4 100 + [M+H] – H2O

80 Poultry Feed Extract 2 H MS → 60 m/z 385

40 HO % Relative Abundance

255.2 287.3 199.3 219.0 325.4 0 150 200 250 300 350 400 450 500 m/z

Figure 34. Full scan MS/MS product ion spectrum from the precursor ion at m/z 385 showing primarily the nonspecific loss of a water molecule

30 Isolation and fragmentation of the ion at match with a similar analysis of a pure m/z 367 yields a full scan MS3 spectrum standard (Figure 35B) and conclusively (Figure 35A) rich in structurally specific confirms the presence of vitamin D3. product ions. This spectrum is an excellent

255.1 100 A Poultry Feed Extract 80 MS3 m/z 385 → 367 → 60 241.2 40 213.1 227.2 285.4 185.0 271.1 311.2 % Relative Abundance 20 325.5 353.2 158.5

0 150 200 250 300 350 400 500 m/z

255.2 100 B Pure Standard 80 Vitamin D3 271.1 MS3 60 213.0 241.3 m/z 385 → 367 → 227.2 40 185.0 285.3 325.5

% Relative Abundance 20 158.6

349.2 367.6 0 150 200 250 300 350 400 500 m/z

Figure 35. Full scan MS3 product ion spectra show much more structural information

31 Environmental Applications Detection of low levels of carbaryl in food Detection of phenylurea herbicides Pesticides in foods and beverages can be a Many of the phenylurea herbicides are very significant route to human exposure. Analysis similar and difficult to distinguish with a UV of the carbamate pesticide carbaryl in extracts detector (Figure 36). Monuron and diuron have of whole food by ion trap LC/MS/MS proved one benzene ring and differ by a single chlorine. more specific than previous analyses by HPLC Chloroxuron has two chlorines and a second fluorescence and single-quadrupole mass benzene ring attached to the first by an oxygen. spectrometry. The protonated carbaryl mole- The UV-Vis spectra are similar for diuron and cule (m/z 202) was detected in full scan mode monuron, but different for chloroxuron. When using positive ion electrospray. A product ion analyzed using electrospray ionization on an LC/MS system, each compound has a uniquely identifiable mass spectrum.

Figure 36. Chromato- gram of phenylurea UV shows class; MS identifies species herbicide with UV and MS spectra 233.1 Norm. Diuron 500 400 199.2 300 Monuron 200

100 291.2 0 Chloroxuron

200 250 300 350 nm 100 200 300 400 500

mAU * 100 80 * * 60 40 20 0

510152025min

32 at m/z 145 (Figure 37) generated by collision- Ion trap analysis was more sensitive than pre- induced dissociation provided confirmation of vious analysis using a single-quadrupole mass carbaryl and was used for subsequent quanti- spectrometer operating in scanning mode and tative analysis. more sensitive than fluorescence detection.

Ion trap LC/MS/MS also confirmed false positives in the 145 m/z +H 100 145 O HPLC fluorescence analysis + caused by a coeluting compound. O C N CH3 H = 202 H M Based on the MS/MS spectrum

80 of the coeluting compound, a Carbaryl possible structure was assigned as shown in Figure 38. HH+ 60 O Abundance O C H N 40 2 4 214 100

N O C NH C2H5 H 86 +H O 20 80 145 + mw 213 [M + H] O 202 + 0 N 60 H H 100 125 150 175 200 225 250 275 86 m/z Abundance O 40 C2H4N

+ Figure 37. Full scan product ion spectrum N O H generated by collision-induced dissociation of the [M + H]+ carbaryl ion at m/z 202 20 145

0 60 80 100 120 140 160180 200 220 m/z

Figure 38. Full scan product ion spectrum of coeluting compound that produced false positives in HPLC fluorescence analysis

33 CE/MS Applications synthetic peptides. When analyzing ppm-levels of analytes in complex matrices, minimal Analysis of peptides using CE/MS/MS sample preparation and short analysis times Capillary electrophoresis (CE) is a powerful enable high throughput. complement to liquid chromatography. Different selectivity and higher chromato- CE/MS with an ion trap mass spectrometer is graphic resolution are its biggest advantages demonstrated using a standard peptide mix. when analyzing clean samples such as The data-dependent acquisition capability of the ion trap triggers MS/MS on the most Intensity intense ion in each 7 [x10 ] TIE, All ± MS scan. Figure 39 1.0 MS shows total ion electro-

0.5 MS/MS pherogram (TIE) and the base peak electrophero- 0.0 gram (BPE). Drops in [x106] the TIE indicate where BPE 200-1350, All MS ± 2 3 4.0 4 MS/MS spectra were acquired automatically. 2.0 1 The averaged mass 0.0 spectrum of peak 4 8.50 9.00 9.50 10.00 10.50 Time [min] (Figure 40) shows a singly charged molecule with m/z of 574.3. Figure 39. Total ion electropherogram (top) and base peak electropherogram (bottom) of a standard peptide mixture This is confirmed as Met-enkephalin (molecular weight 573.7) Intensity by the examination 5 Intensity [x10 ] 5 574.3 of the MS/MS mass [M+H]+ [x10 ] 4 spectrum. The MS/MS 4 574.3 575.2 576.2 spectrum shows all 239.1 0 of the b- and y-series 2 570 574 578 fragments within the 493.1 812.1 1147.2 scan range as well 0 200 400 600 800 1000 [m/z] as fragments from other series. Intensity [x104] b4 6 a4 397.2 425.2 4 b3 y2 b2 278.1 297.1 2 221.0 x2 y3 x3 y4 232.9 262.1 323.1 354.2 380.1 411.1 Figure 40. Full scan mass spec- 0 trum (top) and MS/MS spectrum 225 275 325 375 [m/z] (bottom) of Met-enkephalin from peak 4 of the electropherogram

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Printed in the U.S.A. February 15, 2001 5988-2045EN