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

How has 2020 treated your company? It has been an odd year to say the least, and we are extremely grateful for the opportunities we have had in our field to work alongside and support companies, such as yours, providing medications to help people live the fullest life possible.

While we have been working with our clients on their exciting projects, we have also been working internally on growing! During the past 12 months we have expanded our footprint and capabilities within our current facility as we continue to build trusted partnerships with you – our clients! We have done this by adding the following:

Space

• 3,000 square feet of additional office space
• Three additional laboratories that cover approximately 1,600 square feet
• OHC 3 – 5 lab facility
• A BSL3 lab facility will be completed in the fourth quarter of 2020

Equipment

• Two Agilent 6495 QqQ LC-MS/MS instruments
• Two Agilent 8900 Triple Quad ICP-MS instruments
• Validated Agilent Mass Hunter software
• Two Sciex 6500+ LC-MS/MS instruments
• New Gyrolab xPlore
• Upgraded Watson LIMS to version 7.6.1
• One Agilent Bio-Inert UHPLC

Staff

We have also seen a 15% growth in our staff! We continually look for scientists, lab assistants and techs, IT specialists, software developers, business development/marketing, quality assurance, fellows, and report reviewers for example. We invite you to review our openings on our website and consider joining our role serving the pharmaceutical, biotech, and agrochemical industries, as well as academia.

We look forward to working with you in the months wrapping up 2020 as well as in 2021 with our increased space, resources and staff! Please reach out to us at 610-296-3152 or info@alliancepharmaco.com to discuss how our space, equipment and staff can best meet your drug development and drug metabolism project needs.

Any further questions or need an answer?  Please contact Ryan Klein at rklein@alliancepharmaco.com or 919-801-3146.

Remember when we used to meet in person for our conference experiences? We would gather at conference centers and hotel complexes to attend great seminars, to have insightful conversation, to reconnect with old friends and to make new friends. Everything has changed this year as we all continue to adapt to the COVID-19 pandemic. We understand that many of us are sad about what we may miss, but we are thankful for the technology available that allows us to still attend conferences virtually. We know great interactions, education and conversations can still occur even if it has to happen online!

Our latest blog offers tips on how you can make the most of your virtual conference experience. We hope these tips help you as you adapt to our “new normal”. We look forward to seeing and meeting with you during virtual events in the months ahead. Thank you for the trust you put into us with your drug development and drug metabolism projects!

Tips for attending a virtual conference

We are all used to packing our bags, traveling and preparing for our interactions when it comes to attending conferences. COVID-19, however; has changed all of that this year. As many conferences are turning into virtual events, we thought we’d highlight some ways you can get the most out of your virtual conference experience.

• Prepare mentally for attending the conference. You don't want to just "show up" online. Be prepared with any prep work you would have done prior to an in-person conference. This includes being aware of any potential distractions surrounding you, including your email and phone notifications. It’s tempting to try and multi-task in this circumstance, but do what you can to stay focused on the meeting, just like you would if you attended in person.
  
  
• Be bold. Just as you wouldn't attend an in-person conference and stay in your room the entire time, except for a session or two, you must also be bold in these virtual settings. That means being willing to ask the questions, make the introductions and network. Many virtual conference platforms are offering video chats. Don’t be afraid to ask someone for a face-to-face call.
  
  
• Be active on social media. Social media is a great tool for staying connected and asking questions before, during and after a virtual conference. If you aren't comfortable asking in a live event, use that hashtag to ask your questions on social media channels. Not sure you want your question in public spaces? Follow the social media channels of the conference host, speakers, panelists, sponsors, vendors and attendees, and use those direct message features or find the contact info for that direct email or phone call.
  
  
• Take time to stroll. At a conference, you may pick up a cup of coffee and stroll through the booth areas. Do the same with virtual booth access. Vendors are supporting the virtual meeting through funding and their presence at the meeting. Provide some support for them by visiting the virtual booth and learning about their services in between presentations and during the times dedicated to the virtual exhibit hall.
  
  
• Be patient! Just as you are learning to now navigate a virtual conference, so are all the other attendees, vendors and hosts. There will be times when technology doesn't work right, people forget to mute themselves or even random hiccups occur. Some of it may even feel awkward. Through it all, remember we are all working to adapt in the midst of all the changes COVID-19 has brought this year.
  
  
• Don’t leave early. That last session or final connection may just be the one that makes a difference for you and your company. Make sure to take full advantage of the entire schedule and resist the temptation to head to the couch early.
  
  

You can find our conference schedule on our website and our social media channels. Please remember we may not have a table set up around the corner of the conference hall, but we are here to answer any of your questions and welcome your conversation. Visit us at our virtual booth and chat with our team through the virtual conference links at our scheduled events. You are also always welcome to contact us at 610-296-3152 or info@alliancepharmaco.com.

We look forward to seeing you at that next virtual conference!

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

Seven hundred scientists, students, and exhibitors convened at the Oregon Convention Center in Portland, Oregon, USA this past July 28–31 for ISSX2019.

Alliance Pharma is proud to have presented the three posters below.
Click poster images below to download a PDF file.

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

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.

           The extent of binding to proteins is an important parameter to assess when screening drug candidates during in-vitro ADME studies. Assessing the free fraction of drug available in the biological system is critical to understanding its distribution potential, as it is generally believed that the free fraction drives therapeutic outcomes since it is the free drug (that which is not bound to proteins) that can move to and interact with the sites of action, and high binding to proteins can affect the metabolism and elimination of the drug.

          There are three methods most commonly used to assess protein binding, and it is important to assess their advantages and disadvantages when selecting methods to use for screening studies.

Rapid Equilibrium Dialysis (RED) makes use of a device with two chambers separated by a semipermeable membrane. Spiked matrix is added and over an incubation period, the free drug comes to equilibrium across the membrane while proteins and protein-bound drug cannot move across the membrane; free fraction is calculated by comparing the concentration of drug from the protein and protein-bound drug side to the protein-free, free drug side.

Advantages: Most common method (easy cross-comparisons), simple apparatus/setup and high throughput

Disadvantages: Requires long-term stability in the matrix (typical incubation period is 4 hours), nonspecific binding to the device/membrane, volume shifts and protein leakage over the incubation period.

Ultracentrifugation (UC) uses high-speed centrifugation to precipitate proteins and protein-bound drug from a spiked matrix; free fraction is calculated by comparing the concentration of drug in the protein-free supernatant and the intact spiked matrix.Advantages: Avoids membrane-related issues (nonspecific binding, leakage across membrane, etc.)

Disadvantages: Usually low-throughput, harder to control pH, temperature (conditions that can significantly affect protein binding), requires longer-term stability in matrix (typical centrifugation time is two hours), the supernatant may not be completely protein-free, leading to an artificially high free fraction observed.

Ultrafiltration (UF) uses a two-chambered device separated by a semipermeable filter. Spiked matrix is loaded into the device and centrifuged at moderate speed, during which time free drug passes through the filter while proteins and protein-bound drug cannot; free fraction is calculated by comparing the concentration of drug in the protein-free filtrate and the intact spiked matrix.

Advantages: Suitable for unstable compounds (typical centrifugation time is 30 minutes), quick and technically simple, high throughput/useful to screen many compounds to rank free fraction

Disadvantages: Nonspecific binding to the filter membrane or collection chamber (under-estimation of free fraction), molecular sieving may occur (liquid moves through filter more quickly than the drug, under-estimating free fraction)

          An enzyme-linked immunosorbent assay (ELISA) is a chemical test to detect the presence of proteins or other molecules. The enzyme-labeled antibody binds to antigens leading to a color change that is used to quantify antigen-antibody interactions. Quantification of these concentrations requires measuring the intensity of the color change, which indicates the concentration of antigen bound to the antibody. ELISA’s include four variants: direct, indirect, sandwich, and competitive, which all have their respective advantages and disadvantages.

Benefits of an ELISA

High Sensitivity: The concentration of both antibody and antigen can be quantified on the nanogram scale. This means that scientists can use a smaller amount of enzyme, which still produces a large scale catalytic reaction.

High Specificity: Antibodies are very specific to their targets due to their spatial configuration and chemical structure. They can easily recognize and bind to antigens similar to how a key unlocks a specific door.

Versatility: Indirect ELISA’s allow for multiple primary antibodies created in one species with the same labeled secondary antibody used for detection. Also, it’s easy to wash off any unbound antibodies by applying a detergent solution to the plate.

Clinically approved ELISA tests for diseases like HIV, Lyme disease, and tuberculosis are proving their efficacy and value to the Pharmaceutical industry.

Alliance Pharma is dedicated to bringing comprehensive solutions to our collaborative partnerships.  We understand the importance of efficiency and our team of scientists strive to provide the highest analytical services for your project. 

Our recent purchase of multiple Agilent 6545XT AdvanceBio QTOFs – which is specifically designed with biopharmaceutical characterization in mind - provides high resolution and sensitivity for qualitative and quantitative analyses of biomolecules ranging from peptides to intact monoclonal antibodies. The iterative peptide mapping workflow allows us to dig deep into samples with a wide range of analyte concentrations (i.e. host cell proteins and sequence variants). The Agilent 6545XT AdvanceBio QTOF maximizes our uptime by performing thousands of protein injections without degradation of performance with mass accuracies within 10 ppm for glycosylated intact proteins. 

At Alliance Pharma we believe keeping up with the latest technology helps us to provide the best services for our clients.  The 1290 Infinity II LC system when coupled with the 6545XT, represents the next generation LC for ultrahigh-performance liquid chromatography with superior reliability, offering efficient and reproducible separations of biomolecules for detection by MS or integrated UV and FLD modules. 

By using instruments that complement our scientist’s knowledge and experience we meet analytical objectives, increase productivity, and are cost-effective.  Contact us to see how our recent instrument purchase will help keep your project on track.  Call or email Ryan at 919-801-3146 or  rklein@alliancepharmaco.com for more information.