Tuesday, 31 October 2017

Gradient HPLC: Is your mobile phase causing a ghost peak? Here's an easy way to find out...


Ghost peaks from mobile phase in gradient HPLC
Although not usually a problem in HPLC methods that use isocratic elution, ghost peaks resulting from the mobile phase can be a problem for gradient elution methods. Contaminants present in the mobile phase can behave like sample components and interact with the column during the gradient, leading to a ghost peak (or peaks) in the chromatogram.

However, it is easy to diagnose if your ghost peak is from the mobile phase. Simply run a blank gradient. It’s better if you can do this without performing an injection (to rule out the injection as the source of the ghost peak). In Empower, it is called ‘condition column’. If the peak is coming from the mobile phase then increasing the equilibration time at the beginning of the gradient will increase the size of the peak. For example, you could compare holding at the starting conditions for 5 minutes to holding at 10 minutes. The ghost peak will double in size if it is coming from the mobile phase.

 

Wednesday, 11 October 2017

Validation Challenges for 'Area Percent' Methods

Analytical methods which use area normalisation, or ‘area percent’, as a calibration model are common in the analysis of biomolecules by chromatography and electrophoresis. They present particular challenges when it comes to validation.
The approach is to assign a value of 100% to the area of all the peaks in a chromatogram or electropherogram and then quantify individual peaks as a percentage of that total. Although a very simple idea, the reality is that these methods can be quite complex. In particular, the interpretation of what the percentage value assigned to each peak actually means. If a correlation between this value and the amount of the component present in the sample for analysis is desired then it has to be assumed that the size of the peaks relates to how much is actually there.

This may not actually be the case. For example, if using the popular detector of UV, then the size of the peaks will relate to not just how much is there but also the type of chromophores present in the molecules. Additionally, an area percent approach assumes that there is a linear correlation from zero to 100% which allows each peak to be expressed as a percentage, this may not be the case if the size of the peaks are not within the dynamic linear range of the detector.

When it comes to method validation, it is desirable to address these assumptions and demonstrate that they are justified and this may be achieved if there are reference materials for the components in the sample. However, often the reason for using this approach is that external reference materials for the sample components separated by the method are not available. In this case method validation is challenging.
If you want to learn more about validating methods then you may be interested in the following courses from Mourne Training Services Ltd:

Monday, 11 September 2017

MTS Recommends... UHPLC, Part II: Benefits

UHPLC, Part II: Benefits
Aug 01, 2017
By Michael W. Dong, Davy Guillarme
Volume 35, Issue 8, pg 486–495

"This installment in our series on ultrahigh-pressure liquid chromatography (UHPLC) highlights its benefits in fast analysis, high-resolution separations, high performance liquid chromatography (HPLC) method development, reduced solvent and sample usage, and enhanced sensitivity and precision performance."

MTS Recommends... UHPLC, Part I: Perspectives and Instrumental Features

UHPLC, Part I: Perspectives and Instrumental Features
Jun 01, 2017

By Michael W. Dong
Volume 35, Issue 6, pg 374–381

"This installment highlights historical perspectives on the development of ultrahigh-pressure liquid chromatography (UHPLC) into a modern high performance liquid chromatography (HPLC) platform and describes the important instrumental features common to most commercial equipment."

MTS Recommends... How chemistry started life on Earth

Tuesday, 15 August 2017

MTS Recommends... Analytical Strategy in the Development of Biosimilars

Analytical Strategy in the Development of Biosimilars 

The author outlines an analytical strategy for establishing similarity in biosimilar development and approval.

M. DiPaola, “Analytical Strategy in the Development of Biosimilars," BioPharm International 30 (8) 2017.

Friday, 4 August 2017

Why are 'Bio' Test Methods So Tricky to Validate?

 

Thursday, 3 August 2017

MTS Recommends...

Responding To Regulatory Inspection Observations: Do's and Don'ts 


By Martin Lush, Global VP, NSF Health Sciences, in Outsourced Pharma
"It’s Friday, it’s late, and you are just leaving for the weekend. The inspection you hosted two weeks ago remains a painful memory. The exit meeting didn’t go well — five major observations, all relating to your quality system. When your boss enters your office, you know it’s not to wish you a good weekend. She looks stressed, anxious, and keen to off-load a big problem …"

Thursday, 27 July 2017

HPLC: Should I use a Column Log?


When I deliver my HPLC troubleshooting course, one of the questions that I ask delegates is whether they use a column log. The answer is usually 'yes' from most of the class. Then I ask again do they use the column log (as in reading and reviewing the information in it) and the answer goes down to just a few in each class. The column log can be a great source of useful information if used properly but it can be difficult to get a column log system working effectively.

The benefits of keeping a log for the use of your HPLC columns is that you have a record of what was used on a particular column. This may be used in a number of ways:
  • It could help to predict when a column is coming near the end of its life. This is much easier if the column has only been used for a particular method, or perhaps type of method.
  • It could help to provide information on how this particular column behaves as it nears the end of its life (again easier if it is dedicated to a particular method or type of method). The signs of column degradation are not always the same. Sometimes you might observe that the pressure is increasing and even with the use of guards and filters it is still the criteria that indicates that the column may not be useful for much longer. More often it is chromatography symptoms that will indicate that the lifetime of the column is approaching, such as loss of efficiency and resolution, or peak tailing.
  • You will be able to tell if this individual column is behaving as expected if you have previous ones to compare it to when you are troubleshooting.
  • You will know if you are getting value for money from your column based on how long it lasts.
One of the reasons that it can be difficult to make a column log system work effectively is that it is not always easy to remember to fill them in, or to persuade others to remember to fill them in! Additionally they may be seen as something that is required and done for that reason rather than something which is considered genuinely useful. Thus they may end up filled in but nobody ever looks at them!

I usually advise that it is best to choose carefully when deciding what to record in the log, too many required entries will reduce the likelihood of it being completed. The list below includes many of the useful entries that you could choose from. Probably one of the most important is a running tally of the number of injections that have been performed on the column since this is how we measure lifetime. After that, be pragmatic and choose those that would be most useful for a particular column/method combination. This won't be possible for the first column you use for a particular method but after that it is worth updating the log fields based on experience to make your life easier. 

Typical Column Log Entries Include:
  • Date used
  • Samples injected
  • Method used (if used for more than one method)
  • Number of injections
  • Efficiency, N (not useful for gradient methods)
  • Resolution (may need to specify which peaks)
  • Tailing/fronting (often major peak is used, if applicable)
  • Pressure
  • Retention times (some variation is normal from run to run)
  • HPLC system used
  • etc.


 

Thursday, 6 July 2017

MTS Recommends... Column Care

Column Care for the Long Haul—Considerations for Column Storage


MTS Recommends... Column Care for the Long Haul—Considerations for Column StorageBy Dwight Stoll
LCGC North America
Jul 01, 2017, Volume 35, Issue 7, pg 434–439

"Several factors influence the useful lifetime of high performance liquid chromatography (HPLC) columns. In this installment we consider some of the details associated with preparing a column for storage, with an eye toward choices that will pay dividends in future use of the column."

Monday, 3 July 2017

MTS Recommends ... Data Integrity Expectations of EU GMP Inspectors

Data Integrity Expectations of EU GMP Inspectors


By Orlando Lopez
 "The author reviews key technological expectations of EU GMP inspectors on the integrity of e-records."

Monday, 17 April 2017

HPLC Autosampler Vials: Dos and Don'ts

The HPLC vial is a seemingly minor component of an HPLC system but it has the potential to create major problems. This is particularly true for quantitative analysis. The following list of dos and don’ts will help you to avoid some of the more common pitfalls.

Do:

Choose your vial carefully - There are many different suppliers of HPLC vials, and they each offer different vial options so it is important to choose the vial that is most suitable for your intended use. For example, certified vials may be essential for sensitive trace analysis so that you can be sure that the peaks detected are definitely from your sample and not from the vial. For more routine analysis, certified vials will probably be an unnecessary expense. You may require amber vials for light sensitive sample to maximise lifetime. Vial inserts may help to inject very small volumes of sample. However, many labs like to use the same vial for all HPLC use and not have to choose a vial for each analysis. This is usually a good strategy but during method development (or in the initial method implementation phase if the method is already developed) it is a good idea to consider whether the vial may present any potential problems. The vial cap should also be considered for compatibility with both the sample and the HPLC injector.

Label each vial - You may think that you will remember what is in each of your vials but it is all too easy to forget or have a mix-up. Thus it makes sense to label vials. In a controlled environment (such as a QC lab) it is not just good sense but essential to label vials, to ensure that all analysis performed is fully traceable. There isn’t a lot of writing space on a vial so a clear and easy to decipher naming convention is advisable.

Equilibrate vials to desired temperature
- The amount of sample injected needs to be carefully controlled for quantitative analysis, and the temperature of the sample for injection will influence the amount injected. Therefore, it is important to make sure that sample vials are equilibrated to the desired temperature prior to injection. The ‘desired’ temperature may be the ambient lab temperature, or if a temperature controlled sample compartment in the autosampler is employed, it may be different to the ambient laboratory conditions (usually cooler to prevent solution degradation). In both cases sample vials should be equilibrated prior to injection.


Don’t:

Overfill – If the vial is completely filled to the top then it is likely that the injection volume will not be accurately injected due to the vacuum produced when the sample aliquot is removed. It is advised that the vial is filled to the shoulder to allow a headspace at the top.

Try to inject more than is in the vial – If more than one injection is taken from the same vial (for example, injections of standards) then it is easy to forget that there is a finite amount of solution in the vial. Make sure that the total volume of injections taken does not exceed the capacity of the vial.

Reuse vials – Although it would be desirable to reuse vials, from a cost and waste perspective, it probably isn’t worth the effort of trying to clean and dry vials so that they can be used again. The effort is time-consuming and the risk of contamination is very high.

 

Thursday, 13 April 2017

MTS Recommends... Covalidation Strategies

Covalidation Strategies to Accelerate Analytical Method Transfer for Breakthrough Therapies


Apr 02, 2017
By Kieran O’Connor, Nicola Hulme, Yueer Shi
Pharmaceutical Technology, Volume 41, Issue 4, pg 38–41, 44–48

Abstract:
"An important operation in the registration approval of a new drug product is the commercial-scale validation campaign at a receiving manufacturing site, which requires the technology transfer of analytical methodology from the transferring site. Traditionally, Bristol-Myers Squibb (BMS) used the comparative testing technology transfer model. In a recent launch of a product with breakthrough designation status, however, BMS used the covalidation technology transfer model, which involves simultaneous method validation and receiving site qualification. The authors describe the development and implementation of covalidation strategies to accelerate analytical method transfer using examples from a pilot study."

Monday, 10 April 2017

Wednesday, 5 April 2017

MTS Recommends... The Sound of Compliance

MTS Recommends... Evaluating Progress in Analytical Quality by Design

Evaluating Progress in Analytical Quality by Design

"The authors present the results of a survey of small- and large-molecule pharmaceutical and biopharmaceutical companies on implementation of Analytical quality by design concepts."
 Apr 02, 2017
By Mark Argentine, Kimber Barnett, Marion Chatfield, Elizabeth Hewitt, Patrick Jackson, Shreekant Karmarkar, Ariane Marolewski, Andrea M. Pless, Andy Rignall, David Semin, Mark D. Trone, Qinggang Wang, Zeena Williams, Yanqun Zhao
Volume 41, Issue 4, pg 52–59

Wednesday, 25 January 2017

MTS Recommends... A Statistical Decision System for Out-of-Trend Evaluation

A Statistical Decision System for Out-of-Trend Evaluation


Jan 02, 2017
By Niels Væver Hartvig, Liselotte Kamper
Pharmaceutical Technology
Volume 41, Issue 1, pg 34–43

"The authors present a set of statistical decision rules based on linear regression models that can be implemented in an automated trend system to assist stability studies. The models combine historical stability and analytical method data with data from stability studies, and allow the responsible person to routinely evaluate stability results based on statistical tools, without the need for expert statistical assistance. The system provides a fast and standardized framework for evaluating parameters that approximately follow a linear degradation path or are constant."