LCMS-8040 Triple Quadrupole Liquid Chromatograph Mass Spectrometer (LCMSMS)

Wendy Gavin

LC/MS General Guide

Version 2

July 2017

Table of Contents

1. References…………………………………………………………………………………………………………………. page 3 2. LC/MS Instrument……………………………………………………………………………………………………… page 4 3. Basics of LC/MS………………………………………………………………………………………………………….. page 6 4. Atmospheric Pressure Ionization……………………………………………………………………………….. page 7 a. Electrospray Ionization………………………………………………………………………………….. page 7 b. Atmospheric Chemical Pressure Ionization……………………………………………………. page 9 c. DUIS……………………………………………………………………………………………………………… page 9 5. Triple Quadrupole……………………………………………………………………………………………………… page 11 a. Tandem MS/MS……………………………………………………………………………………………. page 14 b. Isotopes……………………………………………………………………………………………………….. page 16 6. Common ESI adducts…………………………………………………………………………………………………. page 17 7. Liquid Chromatography…………………………………………………………………………………………….. page 19 8. Analysis Modes…………………………………………………………………………………………………………. page 21 9. Sample Prep/ Important Facts ………………………………………………………………………………….. page 26

1. References  LC-MS Training Manual pdf.  Steps for LC-MS pdf.  LCMSMS_OperatorsGuide.pdf.  EEM-07-002-A Level 1 Safety Training.pdf  RES-07-001-ETIC Emergency evacuation.pdf

2. LC/MS Instrument-Shimadzu LC/MS 8040 A. LC Unit

B. Pumps –LC-20AD

Pumps

4 LC-20AD

C. Degasser Unit

Degasser

D. Auto-Sampler

SIL-20AC

Cools samples

3. Sample Preparation  Dissolve sample in acetonitrile, methanol or water. One can use a mixture of these solvents. Do NOT use THF, DMF, DMSO.  Filter through 0.22 micron filter  0.1% v/v, thus 1ml of acid in 1 liter of solvent

4. Basics of LC/MS

Liquid Chromatography Mass Spectrometry (LC/MS) is an analytical technique that combines the separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry. LC/MS is oriented towards specific detection and potential identification of chemicals in the presence of other chemicals (mixture).

Liquid Chromatography is a fundamental technique used to separate a sample into its individual components. The separation occurs based on the interactions of the sample with the mobile phase and stationary phase. Liquid chromatography separates the components of the sample based on differences in their affinity for the stationary phase or mobile phase. LC can separate of wide range of organic compounds from small-molecule metabolites to peptides and proteins.

Traditional detectors for LC include: refractive index, electrochemical, fluorescence, and ultraviolet- visible (UV-Vis). Such detectors quantify substances based on retention time and quantitate substances based on peak intensity and peak area. Chromatography offers great resolution, but quantifying and quantitating substances accurately can be difficult, especially when components elute at the same time.

Mass Spectrometry (MS) offers highly sensitive detection technique that ionizes the sample components using various methods. The resulting ions are separated in vacuum based on their mass-to-charge ratio and the intensity of each ion is measured. MS detectors generate valuable information about molecular weight, structure, identity, quantity, and purity of the sample. MS detectors increase confidence of qualitative and quantitative analyses.

MS is a more sensitive and more specific detector than all other LC detectors. It can analyze compounds without a suitable chromophore. It can identify compounds in unresolved chromatography peaks, reducing the need for perfect chromatography.

LCMS unit consists of a unit for separating components (HPLC), an ion source that ionizes the sample, an electrostatic lens that introduces the generated ions, a mass analyzer that separates ions based on their mass-to-charge ratio, and a detector unit that detects the separated ions.

5. API-Atmospheric Pressure Ionization

The atmospheric pressure ionization unit ionizes the sample sent from the LC under atmospheric conditions.

Ion sources ionize the analyte molecules and separate the resulting ions from the mobile phase. Atmospheric pressure ionization (API) ionizes analyte molecules first, at atmospheric pressure. These analyte ions are mechanically and electrostatically separated from neutral molecules. Ions generated are stripped of solvent, focused into a beam, then delivered to the quadrupole. Electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are two common API techniques. ESI is best suited for ionic compounds of high polarity, where APCI is good for low or medium polarity compounds.

A. ESI

The ESI source operates at atmospheric pressure. The sample solution is sprayed from a small tube into a strong electric field in the presence of nitrogen to assist desolvation. The liquid aerosolizes as it exits the capillary at atmospheric pressure, the desolvating droplets shedding ions that flow into the mass spectrometer, induced by the effects of electrostatic attraction and vacuum. ESI can be used for a wide range of samples such as natural products, proteins, peptides, biological macromolecules and pharmaceuticals.

The principle of ionization for an ESI probe is that the sample solution is drawn into a capillary tube with a high voltage of around 3 to 5 Kv applied to it. Nebulizer gas is blown out around the outside of the capillary tube, spraying the solution and generating fine droplets that are electrostatically charged with the same sign as the applied voltage. During the course of movement, the charged droplets are subject to vaporization of the solvent and they disintegrate when the repulsive force among the charges exceeds the surface tension of the liquid. Through repetition of vaporization and disintegration, very fine droplets are achieved, and ultimately it is thought that sample ions are released in the vapor phase.

The ionization process does not fragment the sample into smaller charged particles. Instead, it turns the sample into smaller droplets which in turn will be further desolvated into even smaller droplets. This creates molecules with attached protons. These protonated and desolvated molecular ions will pass through the mass analyzer to the detector where the mass of the sample can be determined.

ESI allows for the production of multiply charged species. This results in the ability to analyze very high molecular weight species. One sees a distribution of ions for peptides and proteins.

ESI

 Soft ionization technique so that intact molecular ions are observed  Ionization controlled by pH  Introduction of ions to MS requires removal of solvent  Can be applied to larger molecules and forms multiply charged ions  Must contain polar functional groups

Proteins have many suitable sites for protonation as all of the backbone amide nitrogen atoms could be protonated theoretically, as well as certain amino acid side chains such as lysine and arginine which contain primary amine functionalities. ESI can ionize peptides and proteins since they can take on multiple charges. Since the mass spectrometer measures m/z, this brings the apparent mass down to 500 to 2000m/z.

B. APCI

APCI ionizes sample and solvent molecules by spraying the sample solution into a heater using nitrogen. Solvent molecules are ionized by a corona discharge to generate stable reaction ions. Ions are produced in the discharge and extracted to the mass spectrometer. Protons are transferred between these reaction ions and sample molecules by either adding or removing a proton. APCI is used for analyzing highly fat-soluble compounds or compounds that do not ionize in solution.

The solvent gas heated (from 300 to 500°C) inside the APCI probe is ionized on the occurrence of corona discharge due to the application of a high voltage (±3 to 5kV) to the needle. The sample molecules are ionized as a result of ion-molecular reactions (CI reactions) with the solvent ions. Nebulizer gas is used to spray the liquid in the same way as with ESI.

APCI

 Gas phase ionization  Transfer of charge from solvent ion in gas phase  Small molecules and forms singly charged ions  Suited for analysis of non-polar molecules

C. DUIS

Dual Ion Source (DUIS) is an ionization method in which data can be obtained in both ESI and APCI modes. High voltages are applied simultaneously to the nebulizer for ESI and the corona needle for APCI and heated gas is used to assist ionization in the APCI mode. This probe is used as the nebulizer for ESI.

The DUIS ion source adds a corona discharge needle with a voltage of few kV. This latter induces ion formation from low polarity compounds that may not ionize with ESI. This is not a true APCI source.

A. Ion Source Selection

In LCMS, the ionization mode is selected depending on the polarity of the sample. ESI is usually chosen for high-polarity compounds such as those found in drugs and pesticides. APCI is chosen for compounds having lower polarity.

ESI Ionization is achieved by applying a high voltage to the capillary through which a stream of ionized analyte molecules exits in nebulized drop form. In APCI, reaction ions are formed by the electrical discharge around a high voltage corona needle. These ions react in the gas phase with neutral analyte molecules to produce ionization.

For unknown compounds, you can select both APCI and ESI mode using the dual ion source or DUIS. Both ESI and APCI ionization are executed concurrently and continuously without relying on switching between modes. A standard ESI probe is used in the DUIS interface, meaning that uncompromised ESI performance is readily achieved.

The ionization flow using a DUIS is as follows:

1. Sample (including mobile phase) is introduced into the probe. 2. In the ESI probe, the liquid is nebulized into a fine spray. 3. AS drying gas is used to evaporate the nebulized spray, ionization by APCI is produced by the corona needle. 4. Sample ionized by the DUIS passes through the DL to enter the vacuum region.

6. Triple Quadrupole

The mass analyzer used for electrospray ionization is a quadrupole mass spectrometer. A quadrupole mass spectrometer uses four charged rods, two negatively charged and two positively charged, that have alternating AC and DC currents. The rods are connected to both the positive terminal of the DC voltage and the negative terminal. Each pair of rods contains a negatively charged rod and a positively charged rod. The molecular ions are then sped through the chamber between these pairs of oppositely charged rods making use of a potential difference to do so. To maintain charge, and ultimately be readable by the detector, the molecular ions must travel through the quadrupole chamber without touching any of the four charged rods. If a molecular ion does run into one of the rods it will deem it neutral and undetectable.

Quadrupole MS contains four parallel cylindrical metal rods inside a vacuum chamber, positioned equidistant from the center axis, arranged in a square. The analyte ions are directed down the center of the square. Voltages applied to the rods generate electromagnetic fields. The electromagnetic fields determine which mass-to charge ratio of ions can past through the filter.

Quadrupole MS systems can separate ions at lower vacuum levels (10-2 to 10-3 Pa) than other mass separation methods. Thus, when interfaced with LC unit, the drop in vacuum level caused by the interface has minimum effect on mass separation performance. Quadrupole MS has a mass measurement range of up to 2000m/z, which enables qualitative analysis in a practical range of molecular masses. High-speed switching enables simultaneously monitoring multiple selected ions (SIM) for higher-sensitivity simultaneous quantitative analysis of multiple components.

Quadrupole MS can operate in two modes called scanning (scan) mode or selected ion monitoring (SIM) mode. In scan mode, the mass analyzer will monitor for a range of mass-to-charge ratios. Scan mode is good for qualitative and quantitative analysis. In SIM mode, it will monitor for only a few mass-to charge ratios. SIM mode allows quantitative analysis.

The mechanism of analysis for the mass spectrometer is that samples are introduced from the liquid chromatography. ❶ The sample is sprayed and ionized under atmospheric pressure by the atmospheric pressure ionization probe. ❷The ionized sample is introduced through the sample introduction unit or desolvation line (DL), oriented 90° to the spray, into the first stage primary vacuum chamber, where it is efficiently focused at the tip pf the skimmer by the Qarray where multi-stage high frequency ion guides are arranged. ❸The rear section of the skimmer [asses through the high frequency ion guides (multi- poles) arranged in the second and third vacuum chambers. ❹The ions are separated according to their mass-to-charge ratio (m/z) by the quadrupole mass filter with pre-rod and the collision cell and are detected by the detector. ❺The detected ions signals are first amplified by the amplifier and then processed by the LabSolutions data processing software.

The charged droplets that have been spray ionized are heated by the desolvation line (DL). This heating serves to remove the solvent and introduce ions into the vacuum. The lens system is made up of the Qarray, skimmer, multipole and entrance lens. The ions generated under atmospheric pressure are efficiently focused, and then introduced into the quadrupole rods by the lens system.

The vacuum housing comprises the atmospheric pressure chamber and the first, second, third, and fourth vacuum chambers. The rotary pump evacuates the first vacuum chamber and the rear part of the triple inlet turbo molecular pump, while a single inlet turbo molecular pump evacuates the second, third, and fourth vacuum chambers. The pressure is measured by the Pirani gauge fitted to the first vacuum chamber and the ion gauge fitted to the fourth vacuum chamber.

The nitrogen gas supplies 3 types of gas: nebulizer gas, drying gas, and standard sample delivery gas.

A. Tandem Mass Spectrometry (MS/MS)

A mass spectrum tells us the molecular weight; however, we also want to see the fragment ions. Fragments ions provide structural information for the compound of interest. The first mass spectrometer is used to select a single (precursor) mass that is characteristic of a given analyte in a mixture. The mass-selected ions pass through a CID where they are activated to cause them to fall apart to produce fragment (product) ions. The second mass spectrometer is used to separate the fragment ions according to mass. The resulting MS/MS spectrum consists only of product ions from the selected precursor. + + + ɱ1 = precursor ion ɱ1 ɱ2 + ɴ + ɱ2 = product ion

ɴ = neutral loss

+ Product-ion scan (ɱ1 specified)

+ If we perform an MS/MS experiment to find out all of the product ions, ɱ2 that result from the + decomposition of a specified parent ion ɱ1 , and then this is a product-ion scan. It is used to determine structurally significant fragment ions for a selected precursor ion.

+ Precursor-ion scan (ɱ2 specified)

+ A precursor-ion scan experiment tells us all of the possible precursor ions, ɱ1 that decompose to + produce a specified product ion ɱ2 . This is useful when you know that a particular product (fragment)

14 ion mass is characteristic of a class of compounds. You can identify the mixture components that belong to that compound class.

Neutral-loss Scan ( N specified)

This is an MS/MS experiment that looks for all pairs of precursor ions and product ions that differ by a constant neutral loss, N. This is useful when you know that a particular neutral loss mass is characteristic of a class of compounds, and you would like to identify the mixture components that belong to that class of compound.

Multiple Reaction Monitoring (MRM)

Multiple reaction monitoring is the method used by the majority of scientists performing mass spectrometric quantitation. MRM delivers a unique fragment ion that can be monitored and quantified in the midst of a very complicated matrix. MRM plots are very simple, usually containing only a single peak. This characteristic makes the MRM plot ideal for sensitive and specific quantitation. The MRM experiment is accomplished by specifying the parent mass of the compound for MS/MS fragmentation and then specifically monitoring for a single fragment ion. One could think of this operation as the SIM of a fragment ion. The specific experiment in known as a "transition" and can be written (parent mass -> fragment mass) For example 534->375.

CID-Collision Induced Dissociation

To obtain structural information, analyte ions are fragmented by colliding them with neutral gas in CID

B. Isotopes

A mass spectrometer has the ability to distinguish different isotopes. The presence of isotopes gives each fragment a characteristic series of peaks with different intensities. These intensities can be predicted based on the natural abundance of each isotope. An isotope has the same number to protons but a different number of neutrons which results in a different overall mass. A lot of elements have a variety of naturally occurring isotopes, like carbon, bromine and chlorine.

Most Abundant Secondary Abundance/100 atoms of Primary Element Isotope Isotope Isotope Hydrogen 1H 2H 0.015 Carbon 12C 13C 1.080 Nitrogen 14N 15N 0.370 Oxygen 16O 17O 0.040 18O 0.200 Sulfur 32S 33S 0.800 34S 4.400 Chlorine 35Cl 37Cl 32.50 Bromine 79Br 81Br 98.00 28 29 Silicon Si Si 5.100 30Si 3.400

Each isotope will show up as a separate line in a mass spectrum. The Y-axis is relative intensity, so the height of each peak will correspond to the relative abundance of each isotope in the sample. In the example below, the compound contains a Bromine group. It’s isotope of 81Br is two atomic mass units more of the actual mass of the compound. Notice that the peak heights are nearly identical since each isotope is almost 50% naturally occurring.

79Br and 81Br

Another sample is a compound containing a 35Cl. It has a naturally occurring isotope of 37Cl which is two atomic mass units more at 251amu.

35Cl and 37Cl

Another compound is 12C which has a naturally occurring isotope of 13C. The sample below show the exact mass of 325 m/z with a 13C isotope at 326m/z. The Base Peak of this spectrum is 325 and would be labeled M+. and the 13C with an atomic mass of 1 greater would be labeled (M+1)+. The intensity of each isotope peak is proportional to the abundance of the isotope.

13C

7. Common ESI adducts

Adduct ions are prevalent in LC-MS analyses and can come from any number of sources. An adduct ion is any ion formed by adduction of an ionic species to a molecule, ionization polarities (positive or negative ionization).

The commonly observed protonated molecule, [M+H]+ is technically an adduct ion. However, adduct ions which originate from alkali metals, solvents, or other metal species can cause problems when identifying the molecular ion and interpreting mass spectra.

Example of glucose in positive Scan mode [M + Na]+ = 204. Can’t detect [M + H]+ = 181 Molecular weight of glucose=180.26

[M + Na]+ = 204

8. Liquid Chromatography

LCMS API (atmospheric pressure ionization) is used to analyze organic compounds with low to medium polarity or medium to high polarity. Reversed-phase LC mode is best for separating and ionizing these types of compounds. API is used for ionizing compounds with relatively high polarity by spraying the mobile phase into a strong electric field to form a fine aerosol of charged droplets. Therefore, the mobile phase must be able to dissolve polar compounds (water and polar organic solvents) and readily form charged droplets (solvents with low viscosity and volatile salts).

Mobile phase for API and for dissolving sample

 Must be volatile  Must be aprotic  Common choices  Water  Acetonitrile  Methanol  Ethanol  Propanol  Isopropanol  Butanol  Hexane  Cyclohexane  Toluene  Ethyl Acetate

Mobile Phase

. Must be MS grade . Be aware of source limitations on flow rates  On our instrument never go above 1ml/min

Some fundamental mobile phases are listed below.

Ph/Additives for Mobile Phase

 Use low concentration additives  Beware of  Surfactants  Ion pairing reagents   Strong acids/bases (TFA/TEA)

Mobile Phases Suitable for API

Fundamental Mobile Phase Solvents • Alcohols, such as methanol and ethanol • Acetonitrile • Water (pH adjusted, if necessary) pH Adjusting Reagents (volatile, up to about 10 mM) • Acetic acid, formic acid, • Aqueous ammonia (basic) • Ammonium acetate and ammonium formate (buffer solution)

UnUsable Organic Solvents * • DMSO, DMF, THF, acetone, esters, chloroform, benzene, and hexane * If a "fundamental mobile phase solvent" is present, it usually not a problem if the mobile phase contains some of these organic solvents. (However, the ionization effect decreases as the concentration increases.)

Solvents not to be used for LCMS are phosphate buffer solutions and non-polar solvents such as hexane. Phosphate buffer solution can precipitate its non-volatile salts at the interface and potentially cause mechanical damage. Non-polar solvents like hexane contribute very little to ionizing sample molecules using APCI. Analytical conditions using this solvent would need modifications.

Since there are a limited number of mobile phases that are suitable for LCMS, one can change the column as another option. C18(octadecyl) is the standard reverse-phase column, but one could try a C30 to increase retention or a C8 to reduce retention. A phenyl column could increase separation selectivity. Other options for better LC methods are to try isocratic or a different gradient elution.

M + BH+ [M+BH] + M + A- [M+A]-

TIC -Total Ion current chromatogram is a chromatogram this is displayed using all of the detected ions.

MC- Mass chromatogram shows the change in the intensity of m/z over time for a selected ion with a specific mass.

First-Choice Mobile Phases

Mobile Phase A: 0.1 % aqueous formic acid solution • Small molecular weight (M.W. = 46) • Adjustable to low pH levels • Low contamination • Odorless Mobile Phase B: Acetonitrile • Ionization efficiency of ESI is higher than methanol (lower solvent viscosity is better for producing fine droplets).

Caffeine in MeOH

Caffeine in CH3CN

9. Analysis Modes a. MS Analysis 1) MS Analysis is possible using either Q1 or Q3 in the MS analysis mode. Scan analysis and SIM analysis are also possible. High speed scanning at 15,000µ/sec can be performed in Q3 MS mode.

b. MS Scan Mode 1) Scan measurement is performed while scanning the mass ranges once every fixed interval. This method is mainly used for qualitative analysis.

Q1 Scan Reserpine

Q3 Scan Reserpine

21 c. SIM Mode 1) In scan mode, a mass spectrum is obtained continually. In SIM mode, only ions with the target mass are selectively detected. This enables high-sensitivity analysis with no detection time wasted on the detection of ions with masses that are not required. Since the peak height and area are stable, SIM is normally used for quantitative analysis.

Q1 SIM Reserpine

Q3 Sim Reserpine

d. MS/MS Analysis Mode 1) Specific ions are selected in the first quadrupole mass filter (Q1). These selected ions are referred to as precursor ions. The precursor ions are collided with inert gas in the collision cell (collision-induced dissociation, CID) to generate product ions. By measuring the product ions in the second quadrupole mass filter (Q3), we can obtain information regarding the structure of the precursor ions. This analysis method is referred to as MS/MS. Four types of analysis are possible by configuring either scan or SIM analysis for Q1 and Q3.

22 e. Precursor Ion Scan Mode 1) This mode involves performing scanning in Q1, fixing Q3 to a specific m/z, and selectively analyzing the ions generated in the CID. This allows examination of precursor ions with common product ions, and it is suitable for screening ions with common substructures.

f. Product Ion Scan Mode 1) This mode involves fixing Q1 to a specific m/z and performing selective analysis on precursor ions selected in Q3 that were generated in the CID. As product ion spectra can be obtained, this mode is suitable for examining the structure of ions selected in Q1. This mode would be good for determining the amino- acid sequence of peptides and proteins.

23 a.

Product Ion scan

of reserpine=195

g. Neutral Loss Scan Mode 1) This mode involves maintaining the difference in the m/z for analysis in Q1 and Q3 while performing scan analysis. This allows the scanning of ions desorbed from common neutral fragments. As with the precursor ion scan mode, this mode is good for screening ions with common substructures (neutral fragments).

Neutral loss scan of reserpine

h. MRM Scan Mode 1) This mode involves fixing both Q1 and Q3 to a specific m/z and selectively analyzing ions. Precursor ions are selected in the Q1 and product ions that contain structural information generated in the CID are selected in Q3. As both precursor and product ions are specified and monitored, highly selective quantitative analysis that is low in 2) unwanted substances can be achieved compared to SIM measurement. This means this mode is effective in determining the quantities of trace constituents contained in large matrix samples.

10. Sample Preparation-Very Important

LCMS Important Facts

a) Dissolve sample in MeOH or H2O-MeOH or CH3CN-H2O. b) No DMSO, DMF, THF, or hexane c) Centrifuge your samples before loading them into vials. Particulates can clog column. Recommended to double centrifuge. d) Filter through 0.22µm filter.

Mass Spectrometer Important Facts

a. m/z maximum of 2000 Da b. CID gas 230 c. PG vacuum 80-140Pa (96) d. Neb gas 2 or 3(higher flow rate) e. DL-250-300(for higher flow rates) f. Drying gas 15 or 20 (higher flow rate)

Liquid Chromatography Important facts:

a) Never use 100% water for C18 column – bad for column b) Solvent A is always Water (Acid or base) c) Solvent B is always Organic ( MeOH, Acetonitrile) d) Water has 0.1% v/v solution of acid or base

a. Example: 1ml formic acid in 1 liter of H2O.

e) Use H2O from wall unit or LC/MS grade

f) Store column in 50/50 mixture of MeOH/H2O (no acid or base) g) Read about your particular column. h) Never use a flow rate above 1mL/min. i) Samples ionize better in acetonitrile.