Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful technique in the field of bioanalysis, and it plays a critical role in drug development and clinical trials. This method has a well-earned reputation for high selectivity, sensitivity, and specificity for the detection and quantification of low levels of target analytes in complex matrices. The development and validation of a new LC-MS/MS bioassay is a complex and demanding process that involves assessing its performance via analytical characteristics. Even experienced scientists can face pitfalls when developing and validating a new bioassay, so to ensure that the LC-MS/MS method is reliable and accurate, it is essential to validate the method.

Here, we will summarize the eight essential characteristics of LC-MS/MS method validation, then explain why it is important that you work with an experienced contract research organization (CRO) to ensure quality in each of these areas: 

1. Accuracy
2. Precision
3. Specificity
4. Quantification Limit
5. Linearity
6. Recovery
7. Matrix Effect
8. Stability 
   
   

1. Accuracy

Accuracy refers to the difference between the measured value and the true value of the analyte. Scientists assess the accuracy of an LC-MS/MS method by comparing the measured concentration of the analyte in the sample to the known concentration of the analyte in a standard solution. Even small inaccuracies can lead to significant errors in the final concentration of the analyte, resulting in ill-informed decisions and, potentially, the risk of underdosing or overdosing a patient.

2. Precision 

In LC-MS/MS method validation, precision refers to the degree of agreement between the results obtained through multiple measurements of the same sample under the same conditions, and it is assessed by calculating the variability of these results. Precise results are essential for reducing uncertainty in the final concentration of the analyte and ensuring the reproducibility of the method.

3. Specificity

Specificity refers to the ability of the method to accurately measure the target analyte in the presence of other sample components. This can be assessed by analyzing samples that contain the analyte of interest as well as other potentially interfering substances. Specificity is essential in LC-MS/MS method validation because it ensures that the method can accurately measure the analyte of interest without interference from other components in the sample.

4. Quantification Limit

The quantification limit is the lowest concentration of the analyte that can be reliably and accurately measured by the method. This is determined by analyzing samples with decreasing concentrations of the analyte until the signal-to-noise ratio (S:N) reaches a predefined level (20:1 to enable an increased chance it will be suitable). Defining the quantification limit is key because it provides an idea of what sort of sample extraction technique is needed, as well as determining the sensitivity of the method and the lowest concentration that can be reported. These are crucial to ensuring the accuracy of your results.

5. Linearity

Linearity is the ability of the method to produce results that are directly proportional to the analyte concentration over a defined range. In LC-MS/MS method validation, linearity is determined by analyzing samples with increasing concentrations of the analyte and plotting the response against the concentration. Linearity is essential because it ensures that the method can accurately measure a wide range of analyte concentrations. 

6. Recovery

Recovery refers to the ability of the method to accurately measure the analyte in the sample after the sample has undergone extraction or other sample preparation procedures. Recovery is assessed by spiking the sample with a known amount of the analyte and comparing the measured value to the expected value. This process is essential because it determines the accuracy of the method for your specific sample matrix.

7. Matrix Effect

Matrix effect is the interference caused by the sample matrix on the ionization and detection of the analyte. Matrix effect is evaluated by comparing the response of the analyte in the sample matrix to its response in a pure solvent. The method should be able to accurately measure the analyte in the presence of the sample matrix without interference. This is essential because interference from the sample matrix or metabolites can impact the accuracy and precision of the method and cause variations in the analyte concentration. Careful validation is essential for optimizing methods that eliminate or minimize these risks.

8. Stability

Stability is the ability of the analyte to remain stable in the sample matrix under the conditions of storage and processing over time. It is evaluated by analyzing the samples at different time intervals and temperatures and comparing the results, across which the analyte should remain stable. Stability is essential to ensure that the method can provide accurate, reliable and consistent results.

The Key to Quality in All Eight Areas

Developing and validating a new bioassay using LC-MS/MS is a complex process that requires expensive instrumentation, advanced software, and, most importantly, tremendous expertise. Your CRO partner should have a team of scientists with the depth of expertise in LC-MS/MS method development and validation to ensure excellence in each of the eight essential categories listed above. To do this successfully, they should have access to state-of-the-art instrumentation and analytical techniques and be able to guarantee that the bioassay meets all regulatory requirements. That is the way to ensure that the method is carefully optimized and validated for your specific sample matrix, delivering the insights needed to make confident decisions for your program.

In the biopharmaceutical industry, LC-MS/MS assays require accurate, precise, and robust methods developed in the shortest time possible. At the UK lab of Resolian, we have successfully employed a protocol that reaches these goals consistently and efficiently. This achievement has enhanced our capabilities across the Resolian organization: Fordham and Sandwich (U.K.); Malvern, PA (USA); Brisbane (AUS).

Read the guide to discover our powerful approach to developing LC-MS/MS bioassays.

Our advanced scientific infrastructure and world-class experts attract trials from across the globe and meet their needs with the highest standards and efficient regulatory requirements.

Explore the extensive benefits of Australia’s clinical research ecosystem below.

1. GLOBALLY ACCEPTED DATA
   Alliance Pharma has a world-class reputation for quality data, following the highest level of GCP and ICH standards. Data from clinical trials conducted in Australia is widely accepted by all regulatory authorities including FDA and EMA.
   
   
2. EXTEND YOUR BUDGET BY 43.5%
    includes bioanalytics services
    almost halve your clinical trial costs
   
   You can claim R&D cash refund with the Australian Government clinical trial rebate program. As an example, if you spend $200k on eligible R&D, you can receive $87k (43.5%) back from the Australian Tax Office (ATO). This effectively means you could turn $1million dollars into $1.7 million dollars of research spend in just three years.
   
   
3. RAPID START-UP & APPROVAL
    The Australian Regulatory Framework is the fastest in the world for undertaking early phase clinical research trials
    Trials can start in 4-8 weeks compared to 10 months in the US.
    No IND required for clinical trials. Save up to a year in regulatory timelines and considerable costs.
   
   
4. INDUSTRY-LEADING TECHNOLOGY
   Clinical trials are resource-intensive processes requiring the availability of state-of-the-art equipment and facilities for testing and analysis. We have invested heavily in the industry-leading technology platforms to support speed, communications, and regulatory compliance.
   
   
5. GLOBAL CAPABILITIES
   Local relationships, global execution As a global contract research organization – with sites in USA and UK – we can provide internal assay transfer and validation protocols among our global sites.
   
   Harmonized SOPs, policies, and IT systems to facilitate transfer of methods, data, and information enable you to reduce time, risk, and cost.
   
   
6. SEASONAL DIFFERENCES
   Seasonal differences to Europe and US, and time differences (our Australia lab works while you sleep)Seasonal differences between Australia and Europe or the US allow for yearlong participant recruitment for trials involving seasonal illnesses such as flu or allergies and allows researchers to cover seasonal variations in patient recruitment.
   
   
7. HEALTHCARE ENVIRONMENT
   Australian clinical practices and some aspects of its health care system are similar to the United States, United Kingdom and most of Europe.
   
   
8. DIVERSE PARTICIPANT RECRUITMENT
   The willingness of potential participants and their knowledge of clinical trials is another attractive option for sponsors to conduct trials in Australia.
   
   A mixed population of Caucasians and Asian adds diversity to the participant pool and enables ethnicity differences in treatment response to be studied.
   
   

Need Support to Conduct Clinical Trials in Australia?  Contact Alliance!

As a CRO, meeting client expectations can be an ongoing challenge. What can organisations do to make sure their research is as effective and efficient as possible?

In the Autumn 2022 issue of European Pharmaceutical Contractor magazine, the leadership team from Alliance Pharma reviews what CRO organisations can do to make sure their research is as effective and efficient as possible while meeting the ongoing challenge of their customer's expectations.

Patrick Bennett, Jean Pierre Boutrand, and Vito Saccente at Alliance Pharma

Serving clients is the core of a contract research organisation’s (CRO) business. The more comprehensive a CRO’s service offerings, the more likely it is to attract and retain clients. Recent events – like the COVID-19 pandemic – have set in motion industry shifts. Such shifts demand CROs adapt so that they can continue meeting their clients’ evolving needs . . .

READ MORE

Membrane transporters play a critical role in drug-drug interactions (DDIs). Both major types of transporters — efflux and uptake — can affect absorption and drug disposition in the body. Transporters can impact drug safety and efficacy, acting alone or in concert with drug-metabolizing enzymes.

The picture of what is happening in the body gets increasingly murky when patients take more than one drug, which is increasingly common. To obtain insights of how DDIs occur and what their consequences might be, it is vital to conduct in vitro studies and generate the data required to determine whether clinical DDI studies are needed. This is an area where Alliance excels.

In Vitro Assessment of a Transporters’ Potential Role in DDIs

The full picture of a new drug’s DDI potential involves to determine:

• How a new drug is absorbed and eliminated
• How enzymes and transporters contribute to its disposition
• How to characterize the drug’s effects on enzymes and transporters
  
  

Properly designed in vitro transporter studies can support a prediction of whether a DDI through transporters is likely to be clinically relevant, and Alliance has all the tools needed to thoroughly evaluate potential transporter mediated DDIs. We use Caco-2 cells expressing efflux transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), HEK293 cells transfected with uptake or efflux transporters including OATP1B1, OATP1B3, OAT1, OAT3, OCT1, OCT2, MATE1 and MATE2k, MDR1-MDCKII cells, and HEK293 membrane vesicles expressing P-gp, BCRP and BSEP to study transporters, which are outlined in FDA and EMA guidelines.

Helping Clients Solve Problems and Make Decisions

Our clients’ primary need regarding drug transporters is to determine whether their drug candidate is a substrate or inhibitor of a transporter, and if that is clinically relevant. Clients need to know (1) when to do these experiments and (2) how comprehensive the experiments need to be.

(1) In a clinical program, it is crucial to know when to address questions about transporter mediated drug-drug interactions. That decision is driven by the clinical study design and the timing of the in vitro evaluation — which itself can vary based on the therapeutic indications of the investigational drug. For example, if the intended population is likely to use statins, the sponsor should examine their investigational drug’s potential to interact with OATP1B1/1B3 before initiation of clinical studies in patients. Without a question, the co-medications patients are taking factor heavily, so companies must decide how restrictive they want their studies to be. To avoid these transporter studies early on (Phase I), companies can recruit and enroll only healthy volunteers with no current co-meds prescribed to them. However, when a drug is to be administered to the target patient population — patients actually affected by the disease the drug is being developed to treat — it is critical to consider issues such as co-meds, potential renal or other impairments, and the transporters of which the new drug is a substrate and/or inhibitor.

(2) The comprehensiveness of a transporter study is influenced by the relationship between predicted in vivo drug concentrations and the concentrations at which the drug is interacting with the transporter in vitro. For example, if you have a drug that is an inhibitor at a very high concentration, but that concentration will never be reached in the clinic, you might be able to justify forgoing additional testing. (FDA decision trees for this and related questions are available here.) In vivo protein binding and the free fraction of drug in the in vitro system are important data points also to understand and characterize. These are the kinds of decisions Alliance helps our clients make.

The Key Factors in Choosing a Suitable In Vitro Test System

The appropriate model for in vitro testing depends on the desired output. For example, if you need a rapid turnaround but don’t require detailed characterization of a transporter interaction, rapid screening assays are sufficient. For more comprehensive characterization to determine whether to conduct a DDI study, HEK 293 singly transfected cells or the Caco-2 cell line are very useful for characterizing the kinetics of an interaction in detail and can be used to get the output needed. Choosing the right model to deliver the most accurate prediction of how a new drug will behave in vivo is a critical step.

Validating a Model and Experimental Conditions

Know inhibtiors and substrates are used to validate the transporter study models and experimental conditions and characterize assay performance. In other words, the most well-characterized substrates in the literature — substrates that have been studied in vitro and in vivo — are used to establish the assay initially, and then the assay quality is consistently evaluated to determine that the assays are running properly. This approach allows us to build up a history of in house data and continuously assess model performance.

How Alliance Stands Out in This Area

Alliance’s range of models from cell lines to singly transfected systems provides diverse options for our clients and provides rapid turnaround or comprehensive kinetic characterization. With these models and expertise, Alliance Drug Metabolism Services is able to help our clients answer their most challenging and important questions. The bioanalytical support from the strong Bioanalytical teams at Alliance ensure the study data integrity and dependable timelines. Very few of our competitors have this diversity of models up and running routinely, setting us apart.

One unique advantage for Alliance is our in-house expertise using Caco-2 cells. This is a polarized cell line expressing efflux transporters such as P-gp, BCRP and MRP2 that is physiologically similar to human small intestine cells, making it relevant to in vivo studies. The Caco-2 cell model is a valuable tool for evaluating P-gp and BCRP-mediated transport and assessing potential DDIs.

When it comes to DDIs, drug transporters continues to evolve, and new transporters are frequently identified. More and more drug transporters have been found to be clinically relevant. Alliance is always ready to adopt and implement new models when transporters are identified as physiologically relevant to human. We also have the expertise to characterize new transporter study models lacking of regulatory guidance, for example, our recent adoption of a bile salt export pump (BSEP) model, which does not yet appear in FDA guidance.

Alliance’s team members have a unique and deep understanding on how transporter-mediated DDI studies should be designed and executed. We understand how transporters behave, how they interact, and how they can cause clinically relevant DDIs. Whether a new potential drug is at the early discovery stage or the late developmental stage, Alliance can help our clients in the transporter studies. Visit the Alliance website to explore our capabilities and expertise.

The Land O’Lakes conference series is one of the longest running and most revered teaching conferences in the pharmaceutical industry. Over the last two decades, the Land O’Lakes conferences have been developed by the division of pharmacy professional development within the school of pharmacy at the University of Wisconsin-Madison and have an impeccable reputation within the pharmaceutical industry for shedding light on cutting edge topics, bringing in knowledgeable experts and having an intimate setting which lends itself to some fantastic networking opportunities. 

We at Alliance Pharma are pleased to announce that we will be attending the 22nd annual Land O’Lakes conference on Drug Metabolism and Applied Pharmacokinetics on September 9th thru September 12th in Madison, Wisconsin. We are excited and eager to hear from industry leaders on the new advances in drug delivery, action and metabolism. This years’ meeting is titled ‘Found in Translation: Adaptive DMPK Strategies’, and will include topics focused on target mediated drug disposition (TMDD), endogenous biomarkers and more.

Target mediated drug disposition requires that we think outside the box in terms of drug distribution and elimination from the body. TMDD often exhibits nonlinear pharmacokinetics and pharmacodynamics due to the drugs unusually high affinity to a binding site.

Endogenous biomarker research, particularly in the drug transporter area, continues to rapidly evolve. Our biomarker team at Alliance is a highly experienced team with significant expertise in biomarker method development and analysis, yet we are always eager to learn more!

If you’re going to the conference and would like to meet with us, please contact Ryan Klein at rklein@alliancepharmaco.com or 919-801-3146.