LCMS-8040 Triple Quadrupole Liquid Chromatograph Mass Spectrometer (LCMSMS) Wendy Gavin LC/MS General Guide Version 2 July 2017 1 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 2 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 3 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 5 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. 6 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. 7 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. 8 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 9 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. 10