Liquid Chromatography/Mass Spectrometry (LC/MS)
The Department maintains an Analytical Chemistry facility consisting of a Triple Quadrupole Mass Spectrometer (MS) as well as analytical and preparative liquid chromatography (LC) systems. Under the direction of Dr. Jose Tomsig, this facility provides to Department researchers a wide range of capabilities including quantification of small molecules from biological samples, metabolomics, assay development, metabolite identification and peptide analysis. Details of the capabilities of this shared resource are provided below.
Equipment:The main equipment of our facility is a Liquid Chromatography/Mass Spectrometry (LC/MS) instrument consisting of:
- A Shimadzu Prominence UFLC (Ultra Fast Liquid Chromatography) equipped with two pumps for gradient elution; an autosampler that accepts standard vials (up to 70 vials per batch) and 96- and 384-well plates (up to two plates per batch); and a column oven to maintain the column at the desired temperature between 4° C and 85° C (in 1° steps).
- A 4000 QTRAP MS/MS System (Applied Biosystems) equipped with ionization probes for both electrospray ionization and chemical ionization; an external pump for direct infusion studies; and a diverter valve to divert to waste unwanted portions of chromatographic runs. This is a triple quadrupole, unit-resolution machine well-suited for the study of small molecules (MW <1300) and metabolite identification studies.
- A Dell T3400 Workstation running the software Analyst 1.5.1 (MDS Sciex) for the control of the operation of the LC/MS system.
- A Triversa Nanomate Automated Nanoelectro Spray System (Advion), a chip-based electrospray ionization system that functions as an alternative ionization device for the Mass Spectrometer. This system allows the analysis of very small samples because it produces highly ionized samples at exceedingly low flows (nanoliters/min). This system can be used as an infusion device or coupled to the LC system using a post-column split flow device with or without fraction collection. This system is controlled by its own computer that runs the software ChipSoft (Advion) and can also control the LC/MS system.
- Preparative scale: A Waters System that includes a binary HPLC pump (Waters 1525) with manual injector; a UV detector (Waters 2489); and Fraction Collector (Waters Fraction Collector III). This system is controlled by its own computer that runs the software Empower 3 (Waters). This system can be connected to the Mass Spectrometer.
- Analytical scale: Two backup Waters HPLC systems that can be used with UV, turbidimetric, and radioactive (β-) detectors.
Current Capabilities:The LC/MS system has been used during the past 2 years to:
- Quantify natural small molecules (MW <1300) in biological samples. We have developed LC/MS methods for the study of a variety of analytes such as phospholipids (phosphatidic acids, phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylglycerols, and their corresponding lyso forms); sphingolipids (ceramides, dihydroceramides, glucosylceramides and lactosylceramides, sphingomyelins, sphingosines, sphinganines, S1Ps, dihydro S1Ps); cardiolipins; cholesterol; prostaglandins; catecholamines; aldosterone; fatty acids; acetyl and malonyl CoA; inositol; mono, di and tryacylglycerols; lysobisphosphatidic acids; and oxidized phospholipids. In each case we have methods for the detection of the corresponding internal standard, usually a deuterated or 13C-containing form of the natural analyte or a form of the analyte that contains an unnatural (odd number carbon) alkyl group (e.g. fatty acid). The number of metabolites measured simultaneously range from 10 to 100 in a typical experiment.
- Quantify drugs (MW <1300) in biological samples in the context of pharmacokinetics and pharmacodynamics studies. We have developed methods for the study of a large number of drugs. Most of them are drugs that interfere with the metabolism of sphingolipids. In a typical experiment, drugs levels are measured simultaneously with the levels of natural metabolites that the drugs were designed to modify.
- Identify metabolites. These studies are designed to identify the metabolites of the drugs used in the studies mentioned above with the aim of improving the pharmacokinetics of active drugs. To this end, we use the more advances capabilities of the Mass Spectrometer such as Precursor Ion Scans, Neutral Loss Scans in the context of Information Dependent Acquisition (IDA) protocols.