LC/MS refers to liquid chromatography(LC) coupled directly with Mass Spectrometry (MS) via an atmosphere pressure ionization source (API). There are commonly two major types of API sources: the electro-spray ionization (ESI) source and the atmosphere pressure chemical ionization (APCI) source. Although the mass spectrometer in a LC/MS system is essentially a chromatography detector, LC/MS technology literally revolutionized the way we carry out chemical analysis today.
Currently, AxisPharm Labs is equipped with almost all types of Liquid Chromatography Mass Spectrometry LC/MS systems: Single-quadruple, Triple-quadruple, Ion-trap, TOF, Q-TOF, and so on. Our systems are equipped with ESI, APCI, multiple, Duo ESI, Nano sources, etc. for detecting molecules that are normally not detectable. Our Liquid Chromatography Mass Spectrometry LC/MS systems are also equipped with photodiode array (PDA) detector and/or evaporative light scattering detector (ELSD), reflective index detector (RID), charge aerosol detector (CAD) and fluorescence detector (FLD) for challenging detections. The combination of our LC/MS capacity enables our lab to meet wide variety of analytical needs ranging from compound characterization, quality control, and accurate mass measurement to quantitative analysis, structure elucidation, impurity profiling, and metabolite identification and so on.
Samples that require characterization or quality control (QC) are normally run on our single-quadruple or ion-trap systems. The analysis is usually carried out in full scan mode, meaning the mass spectrometer is set to scan over a mass range covering at least 200 Dalton above the sample mass. The lower mass limit in a full can is usually set at 100 or 150 Dalton. The sensitivity of full scan mode is around the low-micromole level, or ~1 µg/ml.
Our generic Liquid Chromatography Mass Spectrometry LC/MS method is a 7-minute gradient run on a 2.1×50 mm 3u C18 column with binary mobile phases. Depending on the polarity of the sample, we usually need to make several runs and adjust the gradient profile to optimize the separation and then only report the best result from the multiple trials. The report always contains a UV trace at custom designated wavelength or a total summation of 190-400 nm, and a TIC (total ion current) trace. Also included in the report are the spectra of the major chromatographic peaks, except the void volume peak. Since the 7-minute run include equilibrium time, our report may or may not include the equilibrium port of the chromatogram depending on whether there is any meaningful peak showing up during that time.
We routinely accept customer designated method to run on our systems. Usually, the customer has to provide the specific column if the method requires a column other than what we have. In some cases, the customer also needs to provide the solvent(s), such as for normal phase and ion-pair runs.
Here’s a step-by-step overview of liquid chromatography mass spectrometry LC-MS bioanalysis:
Sample Preparation: The first step is the extraction and preparation of biological samples (e.g., plasma, urine, tissue) containing the target analytes. Sample preparation techniques, such as protein precipitation, solid-phase extraction (SPE), or liquid-liquid extraction, are employed to isolate the analytes of interest from the complex matrix.
Chromatographic Separation: In LC-MS bioanalysis, the extracted analytes are separated based on their physicochemical properties using liquid chromatography. The sample is injected into a liquid chromatography system, where the analytes interact with the stationary phase (typically a column packed with a specific material) and separate based on their affinity, polarity, or other characteristics.
Ionization: After separation by liquid chromatography, the analytes enter the mass spectrometer. Ionization techniques, such as electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI), are employed to convert the analyte molecules into gas-phase ions. Ionization can be achieved by applying a high voltage to a nebulized solvent containing the analytes.
Mass Analysis: The ions generated in the ionization step are introduced into the mass analyzer of the mass spectrometer. The mass analyzer separates the ions based on their mass-to-charge ratio (m/z). Common types of mass analyzers used in LC-MS bioanalysis include quadrupole, time-of-flight (TOF), and ion trap.
Detection and Quantification: The separated ions are detected and quantified by the mass spectrometer. The mass spectrometer generates mass spectra, which provide information about the molecular weight, fragmentation patterns, and intensity of the ions. The intensity of the ions correlates with the concentration of the analyte in the sample.
Calibration Curve: To determine the concentration of the analyte in the sample, a calibration curve is constructed. Calibration standards containing known concentrations of the analyte are analyzed using the same LC-MS method. The intensity of the analyte in the sample is compared to the calibration curve to quantify the concentration.
Data Analysis: The data generated from the LC-MS analysis is processed and analyzed using specialized software. Data analysis involves peak integration, calibration curve fitting, and quantification of the analyte concentrations. Statistical analysis and quality control measures are applied to ensure the accuracy and reliability of the results.
Validation: LC-MS bioanalytical methods undergo validation to ensure their accuracy, precision, sensitivity, selectivity, and stability. Validation includes assessing parameters such as linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision, and matrix effects.
LC-MS bioanalysis provides high sensitivity, specificity, and selectivity for the analysis of various analytes in biological samples. It is extensively utilized in pharmaceutical research, clinical diagnostics, toxicology studies, and other areas where quantitative measurement of analytes in complex matrices is required.
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