LC-MS/MS Method Development

What Is LC-MS?

(LC) Liquid Chromatography:

Chromatography is a separations method that relies on differences in partitioning behavior between a flowing mobile phase and a stationary phase to separate the the components in a mixture.
A column holds the stationary phase and the mobile phase carries the sample through it.
Sample components that partition strongly into the stationary phase spend a greater amount of time in the column and are separated from components that stay predominantly in the mobile phase and pass through the column faster.

(MS) Mass Spectrometry:

Mass spectrometers use the difference in mass-to-charge ratio (m/z) of ionized compounds to separate them from each other.

Compounds have distinctive fragmentation patterns that provide structural information to specifically detect compounds.

(MS) Mass Spectrometry

Types of Mass Spectrometers

• LC-MS (Single Quadrupole)
• LC-MS/MS (Triple Quadrupoles)
• LC-Q (Ion Traps, Linear Ion Traps)
• LC-Q-TRAPS (Quadrupole Linear Ion Traps)
• LC-TOF-MS (Time-of-Flight)
• MALDI-TOF-MS
• Q-TOF-MS (Quadrupole Time-of-Flight)
• FT-MS (Fourier Transform)
• Others

LC-MS/MS (Triple Quadrupole)

The instrument can be used for quantification and identification of known biological and chemical molecules in complex extracts and mixtures.

The sum of all these instruments represents a multipurpose and efficient MS centre due to its adaptability. Versatility of the facility refers not only to Ionization modes ( ESI, APCI, APPI, ASAP and EI), but also to diversity of separation technologies available like UPC2 (SCF chromatography), UPLC, GC and Ion Chromatography. Therefore a large number of different chemical compounds can be separated and identified in this facility.

LC/MS/MS Advantages

Can be the most sensitive method for protein ID (The smaller the column the more sensitive)
Unambiguous search results (Very few false positives)
Only a single peptide is required for ID
The ability to perform limited homology searches single substitution within a peptide
Able to ID proteins from simple mixtures (~20) such as immunoprecipitates
The data can be searched against EST databases

LC/MS/MS Disadvantages

Time (Both acquisition and analysis)
Chromatography problems of NanoLC

The LC-MS/MS method development can be separated into two parts: sample extraction method development and LC-MS/MS instrument method development.

Sample Extraction Method Development

A sample extraction step may be performed using protein precipitation (PPT), liquid-liquid extraction (LLE), or solid-phase extraction (SPE) methods. PPT is a commonly used sample extraction method, especially in the drug discovery stage. It is a fast, easy-to-apply method for small molecule analysis. The drawback of PPT is that it is not a selective method like LLE or SPE, so matrix effect or ion interference may be observed in chromatograms. LLE is the other widely used sample extraction method. It can be developed as a specific method and can generate clean sample extract solutions.

Another sample extraction method, the SPE method, separates analytes in a chromatographic way and generates a cleaner resultant solution. Extract solution prepared by LLE and SPE methods are more suitable for ultra high performance liquid chromatography (UHPLC) analysis. All three sample extraction methods can be transferred to robotic systems to increase sample preparation efficiency.

LC-MS/MS Instrument Method Development

LC-MS/MS instrument method development requires appropriate settings of LC system and MS system. For LC system setup, mobile phase, LC column, gradient, flow rate, and column temperature are the most commonly adjusted factors based on method development time, analysis run time, and expected number of samples per day.

The purpose of method development is to establish a fast, reliable method which can provide clear resolution of the analyte(s). For MS system setup, ion source parameters, mass transitions monitored in multiple reactions monitoring (MRM), and collision cell parameters are commonly-adjusted factors based on expected analyte concentration and response. The goal of adjustment is to optimize MRM transition response while remaining free of interference. The LC-MS/MS methods developed by our bioanalytical team offer expedited, sensitive, and isotopic-specific results of the analytes. LC-MS/MS method validation is necessary to obtain reliable data during new drug development. A well-developed and validated method is often used from drug development to post-marketing surveillance.

LC-MS Analysis Service

LC-MS Method Development and Validation

LC-MS/MS Method Development

What is preclinical testing?

In the process of preclinical testing of a compound or biological agent into a drug, the compound involved must go through the testing phase. First, we need to identify potential targets that can treat the disease. Then, a variety of compounds or preparations are screened out. Any compound that has shown potential as a drug for the treatment of this disease needs to be tested for toxicity before clinical testing to reduce the possibility of injury. preclinical testing What is the basis of preclinical testing? According to US Food and Drug Administration (FDA) regulations, a series of tests are required before a new drug is approved for use. In the first stage, basic research determines a hypothetical target for the treatment of a certain disease, and then screens small molecules or biological compounds to discover any substance with the potential to treat the disease. Then, a preclinical research phase followed, before which, as described above, the potential toxicity of the compou

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Inventory of the three major in vitro pharmacokinetic research methods

The metabolic properties of a compound are an essential factor in whether or not it can be used as a drug in the clinical setting, so pharmacokinetic studies of newly synthesized compounds are required in drug development. In vitro incubation with liver microsomes, recombinant CYP450 enzyme lines, and in vitro incubation with hepatocytes are some of the more common in vitro drug metabolism methods. 1. In vitro incubation method with liver microsomes The metabolic stability and metabolic phenotypes of candidate compounds in different species of liver microsomes are good predictors of the metabolic properties of compounds in vivo. They are practical tools for evaluating candidate compounds in the pre-development phase of drug development. Liver microsomes include rat liver microsomes, human liver microsomes, canine liver microsomes, monkey liver microsomes, and mouse liver microsomes. In in vitro incubation of the liver, microsomes are the "gold standard" for in vitro d

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Novel Parkinson’s Therapies Possible with New Mouse Model

Parkinson's disease (PD) is a neurodegenerative disorder that is marked by the accumulation of the protein, α-synuclein (αS), into clumps known as Lewy bodies, which diminish neural health. Now, researchers from Brigham and Women's Hospital (BWH) report the development of a mouse model to induce PD-like αS aggregation, leading to resting tremor and abnormal movement control. The mouse responds to L-DOPA, similarly to patients with PD. The team's study (“Abrogating Native α-Synuclein Tetramers in Mice Causes a L-DOPA-Responsive Motor Syndrome Closely Resembling Parkinson’s Disease”) on the use of this transgenic mouse model appears in Neuron . “α-Synuclein (αS) regulates vesicle exocytosis but forms insoluble deposits in PD. Developing disease-modifying therapies requires animal models that reproduce cardinal features of PD. We recently described a previously unrecognized physiological form of αS, α-helical tetramers, and showed that familial PD-causing missense mutati

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