Liquid Chromatography Mass Spectrometry (LC-MS) is a method or technique used commonly for drug analysis, food analysis, and environmental testing. The advantage of LC-MS analysis is that it enables both high throughput and high sensitivity analysis of analytes. LC-MS assay is increasingly used for a variety of compounds leading to accelerated testing and development of drug candidates. Similarly, LC-MS analysis is one of the most commonly used techniques for conducting DMPK studies. Liquid Chromatography Mass Spectrometry combines the physical separation and detection sensitivity capabilities of both platforms.
In other words, the individual abilities of both the LC and MS techniques are synergistically enhanced by their tandem combination. LC-MS analysis works so well because the Liquid Chromatography separates analyte from a complex matrix into distinct components, while the Mass Spectrometry provides the characterization and quantitation for the components with high accuracy. This technique works exceptionally well for the analysis of both synthesized inorganic compounds, as well as organic and biochemical compounds found in complex biological and environmental matrices. For 15+ years, pioneering biotech/pharma companies rely on our LC-MS/MS Analysis experts to drive industry-leading value and turnaround. Our FDA audited LC-MS testing lab always generates reliable and robust data for your pivotal studies. We routinely pilot a wide variety of GLP LC-MS Method Development and Validation projects to meet your critical bioanalysis milestones.
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The LC-MS assay method is used during various stages of the drug development cycle. Most notably, it is used in the following phases:
Over the past couple of decades, high-performance liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) analysis has emerged as one the preferred analytical technique for new drug discovery assays.
Studies range from early lead optimization to dose range finding studies followed by Tox studies. These studies involve primarily plasma and tissue samples of rodent and non-rodent species.
Typically, GLP toxicology studies are performed using fully validated LC-MS methods. Theoretically, this could be accomplished using HPLC with UV and other detection methods like fluorescence, but these detection methods aren’t as selective and require extensive sample preparation. Use of MS detection in multiple reaction monitoring mode (MRM) is quite selective, and therefore drugs can be analyzed in biological matrices with simple cleanup steps. Also, LC-MS assay can be developed relatively faster for a new drug and are generally very robust and reliable.
These studies range from Phase 1 (dose escalation studies like single ascending and multiple ascending dose, healthy volunteer studies) to Phase III, Phase IV and post-marketing studies. Matrices involved in the analysis range from plasma, serum, blood, urine, feces, and tissues from different organs.
Over the last 10-15 years, the LC-MS/MS method has rapidly become the method of choice during clinical trials. At the early stages of clinical development, it is essential to analyze plasma samples from clinical trials for understanding ADME properties of the drug and ensuring data consistency with preclinical PK studies. Thus, LC-MS/MS methods are developed and validated for the drug analysis and testing in human biological fluids.
Here’s how the entire LC-MS assay works:
As the first step, the liquid chromatography is used to separate the proteins, nucleic acids, or other endogenous material in complex biological matrices. There are only minor differences between HPLC (High-Performance Liquid Chromatography) setup with other detectors compared to how HPLC-MS is set up. Among other aspects, these differences include the column length and flow rate. Benefiting from the mass spectrometer, chromatography columns used in LC-MS/MS tests are much shorter than those used in standard HPLC. For example, the regular columns are about 100-300 mm long while those in the LC-MS method are only about 30-50 mm long. Consequentially, the flow rate for LC-MS is slower than the flow rate in the HPLC technique which is 1ml/min. Since the LC-MS combines the advantages of HPLC with mass spectrometry, it can be used for a wider variety of drug analysis as well as environmental testing and food analysis.
The instrument consists of three major components:
First step in the mass spectrometric analysis is the production of gas phase ions of the compound by electron ionization. The molecules that come out of the chromatography column are highly pressurized. Since the mass spec units operate in a vacuum, the continuous flow cannot be detected by the spectrometer. Therefore, the liquid eluted from the chromatography column must be passed through an interface before it can be transferred to the mass spectrometer. The most common interfaces are atmospheric pressure photo-ionization (APPI) systems, atmospheric pressure chemical ionization (APCI) systems, and electrospray ionization (ESI) systems. The liquid that passes through the interface gets nebulized into a spray, after which it is ionized and transferred to the mass spectrometer.
The mass spectrometer measures the mass-to-charge ratio of the ions and then records the relative abundance of each ion type, producing a mass spectrum of the molecule. The equipment displays results in the form of a plot of ion abundance versus mass-to-charge ratio.
Mass spectrometry is a widely used procedure observed as having outstanding sensitivity. Triple-quadrupole mass spectrometry (MS/MS) offers additional benefits due to its selectivity. In mass spectrometry analysis, it is essential to isolate….
LC-MS analysis is now being used for a variety of tests – from drug development to food analysis to environmental testing. Here’s a list of main LC-MS analysis services that NorthEast BioLab provides:
LC-MS/MS analysis plays a critical role in drug and metabolite quantitation analysis, which is an integral part of the drug development process. We develop and validate LC-MS/MS methods in biological fluids including plasma, serum, blood, urine and other matrices for the analysis of drugs, metabolites, and biomarkers.
Biological matrix samples that undergo the LC-MS analysis are typically collected from the following studies:
GLP/GCP compliant LC-MS analysis is conducted on biological matrix samples:
Therapeutic Drug monitoring refers to the practice of measuring certain drugs at pre-defined intervals to evaluate the maintenance of a constant drug concentration within a patient’s bloodstream. This data collected over multiple timepoints helps optimize the dosage levels. Our GCP compliant therapeutic drug monitoring studies utilize LC-MS analysis to track drug levels.
Bioequivalence studies are performed when a formulation is changed to ensure that the expected in vivo biological equivalence of two preparations. Such studies are also required when a generic version of a drug is being developed and required approval from regulatory authorities. Bioavailability refers to the extent and rate to which a drug’s active ingredient is available at its site of action. LC-MS assay is one of the most suited techniques for drug quantitation related to bioavailability and bioequivalence studies.
Validated multi-analyte LC-MS/MS methods are used to study the interaction between different drugs that often need to be used or prescribed together. These LC-MS studies ensure that there are no adverse reactions borne out of this interaction.
Chiral separation is the process for separating racemic compounds into their constituent enantiomers. Conventional LC-MS/MS analysis apparatus can be easily reprogrammed for Chiral separation.
Analyte stability is a crucial parameter in bioanalysis studies. We perform custom LC-MS tests at various time points per your protocol to report analyte stability.
We use state-of-the-art techniques when it comes to method development and sample analysis. Liquid Chromatography-Mass Spectrometry is one such technology that has become increasingly popular in recent years because it combines the capabilities of two different techniques in a very synergistic manner.
For LC-MS bioanalysis, your partner needs to offer the right experience and expertise. Given how time-consuming and painstaking drug development process can be, you need a partner that’s willing to go that extra mile. At NorthEast BioLab, we combine 15+ years of experience in LC-MS method development and validation with deep client empathy to offer a wide variety of drug discovery and development studies.
Finally, we whole-heartedly cooperate with you to accomplish our shared goals and accelerate your study timeline. Our team always keep the lines of communication open between our clients and frontline managers and lab staff. We bring together our core strengths- operational excellence, regulatory expertise, and scientific experience, to make sure your costs and resources are optimized.
We have 15+ years of experience in performing robust LC-MS method development using appropriate chromatography column and mass spectrometer system settings. Generally, it takes a few iterations to achieve desired LC-MS method parameters before finalizing an effective assay. We must optimize these numerous LC-MS method development parameters, such as ionization mode, parent and fragment transition ion pairs, polarity, and source parameters including gas flows and temperature, to detect and quantitate a chosen compound successfully. First, we must determine how the test article ionizes when developing a mass spectrometry method. Electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) are two of the most widely used methods that can be run in both positive or negative polarities. Our team identifies the selection of proper ionization mode as critical and usually the first condition to be determined. If no prior references in literature are available, our experienced scientists can make an educated and informed guess about initial conditions based on your drug compound details (structure, molecular weight, etc.). Otherwise, we choose an ionization mode and polarity by testing differing ionization probes in both positive and negative polarities to determine the ideal starting approach. Next, our scientists evaluate the MS response and create a methodology that provides the best MS conditions for detecting your compound. Thus, we select the most reliable ionization potential and obtain the most suitable m/z value (parent or daughter ion) for optimal sensitivity and selectivity. Ideally, we could see our analyte(s) of interest distinctly from all endogenous interferences and drug metabolites using robust chromatography and the superior selectivity of MS/MS detection. Typically, the most commonly adjusted factors for LC system setup are mobile phase, stationary phase, or column, chemistry, gradient, flow rate, and column temperature with development tailored to intended use inclusive of method development time, analysis run-time, expected number of samples per day, and analytical concentration range. For the liquid chromatography development, one of the first steps during the LC-MS/MS method development is determining the retention characteristics of the compound(s) of interest. With biological matrices, it is often critical to ensure that test compounds elute away from the solvent front where interference from endogenous sample components can occur. Often, we can quickly solve this puzzle by screening at low and high pH using a simple reversed-phase gradient and a high retention column such as C18, or other suitable stationary phases dependent upon the test article chemistry. Notably, proper sample preparation for LC-MS analysis is critical for ensuring that our assays meet recommended compliance and performance standards. Ultimately, we understand that analytical performance is dependent on a clean and stable sample, yielding method robustness, reliability, and reproducibility.
LC-MS assay offers a sensitive, selective, and reliable approach for analyzing diverse molecules. Different mass spectrometer models provide advantages and disadvantages for viable LC-MS analysis over mass ranges and detection limits. Most biomolecules utilized as reference materials for drugs, metabolites, or endogenous biomarkers, including drugs, peptides, nucleic acids, and other endogenous or synthesized molecules, can be assayed using triple quadrupole mass spectrometry. Triple quads, however, have limited mass ranges, and intact proteins and large biomolecules can alternatively be assayed by other LC-MS testing techniques, including time-of-flight (TOF) or other high-resolution mass spectrometers (HR-MS). Inductively coupled plasma mass spectroscopy (ICP-MS) is especially useful for metallic ion assay in environmental chemistry and clinical toxicology.
In LC-MS lab setting, aqueous and organic solvents, dosing vehicles, or other drug formulations prepared for in vivo or in vitro testing, and biological fluids and tissues are routinely quantified for drug substances, metabolites, and biomarkers. Biological samples for LC-MS analysis may include Blood, Plasma, Serum, Tissue Homogenates/Extracts (Liver, Lung, Kidney, Brain, Tumor, Arterial, Tendon, Skin, etc.), Translucent Matrices (Urine, Cerebrospinal, Tears, Synovial, Aqueous, etc.), Cell Lysates, and several other body fluids or membranes.
For solid tissue samples, sample homogenization is typically performed as part of the sample processing for LC-MS testing. For all matrices, samples must be extracted into solvents suitable for injection for LC-MS assay. Protein precipitation (PPT), liquid-liquid extraction (LLE), and solid-phase extraction (SPE) are the most commonly used sample extraction techniques used in LC-MS labs during sample preparation for LC-MS analysis. The extraction method employed is verified during LC-MS/MS method development and method validation.
In any LC-MS testing method, the required sample volume depends on multiple factors such as calibration range, compound sensitivity, chromatography column, matrix-based suppression or enhancement, mobile phase selection, and detector linearity. Often, LC-MS method sample volume requirement can vary between 2 µL-200 µL. We can use specialized systems such as capillary flow for shallow volumes. Our scientists recommend collecting additional sample volume in pharmacokinetic/toxicokinetic assessments from biological samples to complete reassay or incurred sample reanalysis (ISR) to support your LC-MS method validation.
Buffers and mobile phase solvents used in LC-MS applications should ideally only contain volatile components. The most common volatile salts for buffering in LC-MS mobile phases are ammonium formate and ammonium acetate. Here, adjustments in pH are performed with volatile acids and bases such as formic acid and ammonia. Usually, some non-volatile components get introduced during assays of biological samples with sample injections. Mass spectrometer instruments can handle small amounts of these non-volatile components with routine cleaning and maintenance.
In LC-MS assays, a mechanical region in the flow path is often the source of carryover. Most commonly, carryover occurs in the autosampler needle or chromatographic column used for the assay. Lab technicians can inject blanks during LC-MS analysis to gradually dilute the hold-up of partial samples, resulting in less carryover with each pass. Similarly, we can apply an organic solvent as a needle wash solution when analyzing hydrophobic samples to minimize carryover by solubilizing and washing away the adsorbed sample components. Here, the analyst must handle the needle wash solution in the same way as the mobile selection during the method development of LC-MS assay. Advanced instrument models allow for more solvents, higher flow rates, and other advanced washing mechanisms to help reduce autosampler-based carryover. We can minimize chromatographic carryover by sample dilution, smaller injection volumes, or changes in the stationary and mobile phases of the assay.
Pre-method or Test validation is a process designed to confirm that the procedure employed for a specific assay is suitable for its intended purpose ahead of actual LC-MS method validation. Test method validation provides an overall understanding of the uncertainty of the LC-MS method, stability of the analyte during storage, method processing and analysis, and reproducibility and ruggedness of the LC-MS assay. FDA and other regulatory agencies mandate full method validation for each test article, sample matrix, analyst, and LC-MS instrument system before regulated sample analysis.
We can compare replicate LC-MS/MS runs using experimental duplicate to inform us on precision further and help assess whether our LC-MS method and sample analysis are performing as expected. Generally, replicate samples are analyzed on an as-needed basis for LC-MS testing due to resource constraints and relatively longer run times associated with chromatographic assays.
All high-Quality LC-MS method validations ensure that the analyte of interest can be detected selectively, accurately, and precisely in an appropriate calibration range suitable for sample analysis. Furthermore, these LC-MS method validations must include proper safeguards to verify all test compounds are stable during sample collection, storage, and processing. To that end, FDA guidelines on chromatographic method validation mandate demonstrating selectivity, specificity, sensitivity, accuracy, precision, recovery, and stability under storage and test conditions. Usually, a change in analyte, detection range, or sample matrix results in a shift in the method and the need for additional LC-MS method validation. Frequently, LC-MS assays are validated on any system and for any analyst performing the LC-MS method to ensure that LC-MS/MS analysis results are robust, reliable, and reproducible.
For many molecules, methods for UV/Vis detection may be modified for detection by mass spectroscopy. Often, LC-MS analysis will offer an improved selectivity, sensitivity, and linear dynamic range relative to LC-UV/Vis analysis. Modifications to the method typically include switching to volatile mobile phases for compatibility with MS source introduction and selecting a more appropriate internal standard, such as a stable-labeled isotope of the analyte, for LC-MS method development and validation as well as during sample quantitation.
LC-MS analysis has extensive industrial applications beyond sample quantitation as often LC-MS analytes can be chemically, thermally, or enzymatically labile. For instance, we can perform LC-MS analysis to detect, isolate, or purify certain chemical compounds. In pharmacology and environmental settings, we can utilize LC-MS/MS to assess the stability of xenobiotics, drugs, metabolites, or toxicants in various sample matrices. Furthermore, we could use LC-MS detection for metabolic lability, structural and metabolite identification from in vitro sample incubations or in vivo pharmacology studies, and other in vitro ADME determinations such as protein binding, cell permeability, or drug-drug interaction potential. LC-MS analysis can apply to samples originating from industrial hygiene monitoring, environmental sampling, fermentation broths, growth media, the agricultural industry, process control, biomedical studies, food testing, and countless others avenues.
Generally, triple-quadrupole mass spectrometers are used in analytical and bioanalytical LC-MS laboratories for quantitative small-molecule applications due to their selectivity and sensitivity advantages. Time-of-Flight (TOF) and QTrap LC-MS models are more commonly used in qualitative applications, such as metabolite or structural identification, combined with alternate analytical approaches such as nuclear magnetic resonance (NMR). Laser desorption mass spectrometers (MALDI and SELDI) can be used to analyze large molecular weight biomolecules. Inductively coupled plasma mass spectroscopy (ICP-MS) is utilized to study metallic ions, often in environmental labs and toxicology settings to diagnose heavy metal or radioisotope exposure.
The terms LC-MS and LC-MS/MS are often used interchangeably. Technically, LC-MS refers to a single quadrupole system with only one mass filtering quadrupole, while LC-MS/MS indicates a triple-quadrupole system with two mass filtering quadrupoles. In LC-MS/MS system, Q1 and Q3 quadrupoles function as mass filters and Q2 acts as a collision cell to generate product ions. Typically, LC-MS is more sensitive, but the improved LC-MS/MS selectivity allows for cleaner, more reliable analyses from complex matrices. LC-MS analysis of low molecular weight compounds requires a clean sample matrix to avoid the interference of unwanted ions. We can assay these compounds more efficiently using triple-quadrupole mass spectroscopy, allowing lowered noise, higher selectivity, and increased linear range.
LC-MS analysis offers a sensitive, selective, and moderate-to-high throughput technique for sample quantitation of drugs, metabolites, and biomarkers in samples from various analytical and biological matrices. Different instrument models and detection techniques like ion traps and time of flight mass spectrometers provide valuable tools for qualitative structural identification and drug metabolism studies. LC-MS has historically had limited application for quantitation of intact biomolecules, even as some newer instrument models offer high mass detection. LC-MS lab systems are costly to implement and maintain, require skilled operators for method development, regular instrument calibration and troubleshooting, and provide low throughput for LC-MS sample analysis. That said, clinical laboratories during the last 15 years have experienced an enormous increase in the use of liquid chromatography-tandem mass spectrometry (LS-MS/MS). Newborn Biochemical Genetics and Drug/Toxicology Endocrine laboratories have surpassed screening laboratories as the leading clinical LC-MS/MS users. LC-MS methods now measure most steroids and biogenic amines in US reference/referral laboratories, and the technique is beginning to spread to smaller labs.
LC-MS/MS platform offers higher throughput than gas chromatography-mass spectrometry (GC-MS) and analytical specificity superior or comparable to immunoassays. However, LC-MS/MS has numerous limitations, most of which revolve around the interacting triangle of sensitivity, specificity, and throughput. While sample throughput is better than traditional HPLC or GC-MS, it still falls short of automated immunoassays. Direct sample injection, LC-multiplexing, and sample multiplexing are all evolving methods for increasing LC-MS throughput. Sample cleaning and chromatography optimization are two ways to further improve specificity and sensitivity by avoiding interferences and ion suppression caused by sample matrix components. Additional advantages may be available to the LC-MS platform with next-generation instruments. Among other matters, peptide/protein analysis is the next barrier for clinical LC-MS/MS. The search for multi-biomarker profiles for various diseases has largely failed by LC-MS so far. Still, targeted peptide and protein testing by LC-MS/MS, directed at analytical and clinical questions that need answering, is proving extremely effective. We anticipate that clinical protein/peptide LC-MS/MS will grow at a similar rate to low molecular weight applications.
