Tuesday, 13 December 2011

Ireland HPLC Training Schedule for 2012

The dates for the next MTS open enrolment training courses in Ireland in 2012 are now available:

Tuesday 5th June
How to Run HPLC Methods

Wednesday 6th June
How to Troubleshoot HPLC

Thursday 7th June
How to Develop HPLC Methods

Friday 8th June
How to Develop HPLC Methods for Challenging Separations

We also plan to run the course 'Validation of Analytical Methods for Pharmaceutical Analysis' on Tuesday 23rd & Wednesday 24th October.

The location is Dublin; full details will be on the website early next year. Contact us for more information.

Monday, 12 December 2011

MTS Recommends... Measuring pKa using UV/Vis

When delivering HPLC method development training, I usually advise that the pKa of an analyte is taken into consideration, if appropriate, when selecting a suitable buffer pH. The problem with this approach is that the pKa value is not always known. A resource which may prove helpful is provided in the Chemistry Resources section of the Chemagination website where there are directions on ‘How to measure pKa by UV-vis spectrophotometry’.

Friday, 9 December 2011

Help on: Acceptable Variation for HPLC Retention Time

MTS HELPDESK
Do you have any problems relating to analytical chemistry for pharmaceuticals or training? Send your questions to the MTS helpdesk using our contact form.

Question:
“I assess assay results for imported pharmaceutical products performed by a third party lab. The methods we use do not always specify an "allowed" retention time range. What would a reasonable guide be when assessing this – plus or minus 10%? Basically, what I should like to know is when should we ask the laboratory to investigate/explain a retention time shift from one assay to the next? Put another way, when should the laboratory make system adjustments like the temperature of the column, flow rate etc. in order for the retention time to conform? Often the method will say the active elutes at say 10 minutes. Does this mean that 9 to 11 minutes is also OK, or are there justifiable limits that we can impose?”

Answer:
“The reasons for variation of retention time from analysis to analysis includes small differences in the following: the composition of the mobile phase (in terms of the aqueous and organic solvent portions); the pH of the mobile phase (particularly important for ionisable molecules like acids and bases); HPLC columns (column to column variation), and HPLC systems (particularly the system volume). As a result of these variables it is expected that the retention time will vary and usually small variations do not cause any problems. The potential problems associated with retention time variation from run to run (not injection to injection in the same run, that is a more serious problem which may indicate a problem) includes:
  • The change in runtime means that a peak is not fully collected by the end of the run and thus the analysis has to be repeated.
  • For analysis of mixtures, such as impurity analysis, peaks may be difficult to assign if the retention time varies.
  • Automatic integration events may have problems relating to identifying peaks correctly, thus requiring extra time for reprocessing.
To answer your question, it is difficult to set an allowable retention time range without a good understanding of the individual method. Ideally the person who develops the method should investigate its robustness and thus gain information on the range of retention times which might be expected due to normal variation and where the method still performs as required. Of course, it is not an ideal world and often you will be presented with data from methods where this has not been done, or if it has, it is not written into the method. From experience I would say that about 10 to 20% of the time quoted in the method is a reasonable guide but be careful not to apply it too restrictively. If you have an analysis where the retention time is on the limits, the best way to assess if there is a problem is to look at the chromatograms for the analysis and compare to a previous satisfactory analysis (preferably an example chromatogram should be included in the method). If there aren’t any differences except for retention time, and the system suitability testing is all fine, then the results should be satisfactory to use. If the retention time is very different to that expected and well outside of this approximate range then I would suspect that something has been changed in the way the method was applied and would want the analyst to investigate further.

Although pharmacopoeia sometimes include adjustments to temperature, flow rate etc. to make sure that a peak is at a particular retention time these changes are actually deviations to the method and unless you are following a pharmacopoeia method or have validated your method to show that these alterations are valid then it is not a good idea to change the method parameters.”

Monday, 5 December 2011

New Associate @ NSF DBA

I am delighted to announce that I am now an associate of NSF DBA. You will probably be familiar with NSF DBA, formerly David Begg Associates, if you work in the pharmaceutical industry since they are Europe’s largest provider of pharmaceutical training, both in-house and external, and also provide pharmaceutical auditing and consulting services. I will be contributing my expertise in the area of analytical chemistry, and in particular the analysis and testing section of Qualified Person (QP) training.

Friday, 2 December 2011

HPLC Training in Helsinki

I will be delivering 3 HPLC training courses from the ‘How to...’ series in Helsinki at the end of January 2012 in collaboration with Phenomenex. The courses are:

Tuesday 31st January
How to Troubleshoot HPLC
Wednesday 1st February
How to Develop HPLC Methods
Thursday 2nd February
How to Develop HPLC Methods for Challenging Separations

Contact the Nordic Phenomenex office for more details: nordicinfo@phenomenex.com

Thursday, 24 November 2011

USP Chromatographic Columns

PEAK SOLUTIONS
A resource for chromatographers

If you have ever followed an HPLC method in the United States Pharmacopeia, then you have probably already encountered the problem of selecting a suitable HPLC column for the analysis. Columns are designated by a letter and number which identifies the stationary phase, e.g. L1 refers to ‘Octadecylsilane chemically bonded to porous silica or ceramic micro-particles, 1.5 to 10 μm in diameter, or a monolithic rod’. Unfortunately there are hundreds of columns that fit this description and due to selectivity differences they may not all give similar results.

The outcome of the USP Working group on HPLC Columns is that you can now look up the chromatographic column which was used to validate the procedure. The free online database provides a cumulative listing of columns referenced in gas- and liquid-chromatographic methods related to revisions made to USP–NF since January 1980. USP Chromatographic Columns can be accessed via the USP website, you will need to register to access the database, then search for the monograph in question. Once you know the actual column which was used for the method you can either use this or an equivalent. The USP Column Database provides a tool which can be used to find equivalent columns.

Friday, 11 November 2011

MTS Recommends... ‘HPLC Columns’ by Uwe Neue

‘HPLC Columns - Theory, Technology, and Practice’ by Uwe D. Neue, Wiley- VCH, 1997

Although this book was published some time ago in 1997, it is still a fantastic resource for chromatographers. Neue managed to combine a thorough overview of the theoretical background relating to column technology with a practical guide, all in a very readable style.

Wednesday, 9 November 2011

Help on: Assessing Peak Purity Data

MTS HELPDESK
Do you have any problems relating to analytical chemistry for pharmaceuticals or training? Send your questions to the MTS helpdesk using our contact form.
Question:
“I am developing a HPLC indicating assay for a hormonal drug. I am using the peak purity option to analysis the purity of my peak once the sample has been subjected to 20% degradation under various conditions. The purity results show that my peak is not pure although its 5 overlaid spectrums are more or less identical. The resolution of my drug from its degradation products is greater than 2. I need to know how I can accurately interpret my peak purity results in order to ensure that no other degradation product is co-eluting at the same time as my main peak. I would appreciate any suggestions with regards to this matter.”
Answer:
"I assume that the results you are referring to are from the software that you are using for peak purity analysis. The purpose of this software is to compare the spectra obtained at time points across the peak and detect differences which cannot be observed by eye. Therefore it is possible that there are spectral differences which may be explained by the presence of another peak eluting at a similar time as your drug. When a drug is degraded, the degradation products are often very similar in structure leading to similar retention times and UV spectra. This means that the spectral differences observed during peak purity analysis may be very small.
To investigate whether your purity result is actually due to another peak or is just a result of the way the spectra have been compared by the software requires knowledge of how the purity result was generated. I recommend that you read through any available information on how the software compares the spectra.
Things to consider:
  • What reference spectrum is being used, particularly important if it is a gradient method;
  • Compare the peak purity plot observed for your forced deg sample with that for a pure reference standard;
  • Loss of linearity can result in spectral differences which are not due to another peak, try diluting your sample and compare the peak purity plot obtained with the original.
A final note, peak purity can never be proven. You can only gain confidence that no other peaks have been detected. This is because the UV spectrum for similar compounds may be identical, also if the apex of both co-eluting peaks is at the same time then the spectra across the peak are likely to be identical.”

Thursday, 3 November 2011

Free HPLC Calculator for Method Development

PEAK SOLUTIONS
A resource for chromatographers

The MTS free HPLC calculator now includes a method development tool for HPLC. The tool is based on a scouting gradient approach for determining suitable initial conditions for a mixture of analytes, where a single gradient analysis is used to decide whether isocratic or gradient elution is most suitable, and to select promising mobile phase composition. This will be familiar to delegates on MTS training courses and you should be able to start using the calculator straightaway. I plan to post more details in the new year on how to use the tool for those of you who have not attended one of my courses.

Monday, 31 October 2011

HPLC Training in Athlone

MTS hosted three days of HPLC training last week in Athlone, Ireland. Thank you to all delegates who attended for a great event and for all the feedback on the training. Don’t forget that you can upgrade your certificate of attendance to a certificate of training by completing the online training assessment, the directions are in your course handout.

Get in touch if you have any problems with the assessment or indeed any questions which came up when you applied your new skills back at work. If you missed these courses then contact us, we plan to run some more in 2012.

Thursday, 13 October 2011

Separation Science Europe 2011

Earlier this week, I presented "A Comparison of Recommended Strategies for Reversed Phase HPLC Method Development" at the Separation Science Europe 2011 Conference. The venue for the conference was the Royal institute in London. It was amazing to present at the same location as eminent scientists from the past such as Michael Faraday and Humphry Davy. The conference programme included a fascinating mix of topics relating to LC and GC applied to a wide range of analytes in the pharmaceutical, environmental and bioclinical sectors.

The highlight for me was a presentation from Ron Majors about ‘Just Enough’ sample preparation where Ron described how analysis by LC-MS/MS and GC-MS/MS has enabled the use of simplified sample preparation procedures. Four examples were presented: Protein precipitation in blood plasma; QuEChERS for pesticide residues; Dried Blood (Matrix) Spotting; and Pharmaceutical pollutants in river water with almost no sample preparation. The potential of reducing both time and error by reducing sample preparation is very appealing.

Friday, 23 September 2011

Help on: Problem with the first injection from HPLC vials

MTS HELPDESK
Do you have any problems relating to analytical chemistry for pharmaceuticals or training? Send your questions to the MTS helpdesk using our contact form.

Question: “When I use a single vial for a series of standard injections the area for the first injection is often lower than that for the subsequent injections. What could cause this?"

Answer:
“There are a variety of potential reasons for injector repeatability problems but the most likely explanation when the first injection from an individual vial gives a lower response than subsequent injections is that the vial was overfilled. This creates a vacuum when the needle pierces the cap and withdraws the test solution and thus the syringe does not withdraw the correct amount. On the next injection the vacuum effect is no longer present and thus the syringe withdraws the correct amount. To prevent the problem, take care to leave a small headspace when filling a vial rather than completely filling the vial.”

Thursday, 22 September 2011

Separation Science Europe 2011

I will be presenting "A Comparison of Recommended Strategies for Reversed Phase HPLC Method Development" at the Separation Science Europe 2011 Conference, on the 10th October.

Abstract:
The benefits of using a strategic approach for developing HPLC methods are easily apparent. The numerous possible chromatographic parameters in a typical HPLC method make choosing the most suitable ones for a particular separation very daunting. In particular, how to select one column from the hundreds available? There are a number of different strategies which can be applied, these include: trial and error, changing one variable at a time; finding a method in the literature or finding a method in the literature for a similar compound; and sophisticated column screening experiments combined with computer modelling, peak tracking methods, experimental design and column comparison tools. In this presentation, current recommended method development strategies are reviewed and compared to give delegates an appreciation of the types of strategies which may be applied, so that they can identify the one which is most applicable for their method development needs.

Tuesday, 20 September 2011

How to Troubleshoot HPLC

MTS will continue to work with Phenomenex in the delivery of HPLC training seminars in the UK with a series of troubleshooting courses in November. The dates and locations are as follows:

8th November - Edinburgh
9th November - Crewe
15th November - London

Course price is £195 + VAT per delegate. The price includes: Full day training (including post training assessment), course literature, technical brochures, lunch and refreshments.

If you are interested in attending, contact Phenomenex:
Tel: UK: 01625 501367 or email: ukinfo@phenomenex.com

Thursday, 25 August 2011

HPLC Calculator for Void Volume

PEAK SOLUTIONS
A resource for chromatographers
The MTS HPLC calculator now includes calculation of void volume. This calculation may be useful when multiples of column volumes are required, e.g. for equilibration, and also in HPLC method development to estimate t0, the time in the chromatogram when unretained compounds, e.g. the solvent front, will elute.

Tuesday, 23 August 2011

5 Step Strategy for HPLC Method Development

The 5 step strategy for HPLC method development which is taught in MTS training courses is now available in a summarised format as a free download. Click on the picture to download this useful guide to the important steps in HPLC method development. It is designed as an A5 postcard but will also print well on an A4 sheet.

Friday, 22 July 2011

BARQA Annual Conference 2011

I have been invited to speak on the topic of analytical method validation at the BARQA Annual Conference as part of the session 'Crossing the Plimsoll Line' on 28th September. The title of the presentation is Analytical method validation: Establishing that test methods are 'fit for purpose'. The full programme for the conference can be accessed on the BARQA website.

Tuesday, 12 July 2011

How to Become an Expert on HPLC

HPLC Training Courses from Mourne Training Services
October 2011 - Radisson Blu Hotel, Athlone, Ireland
Mourne Training Services will deliver three training courses on HPLC topics in Ireland during October, which together provide comprehensive instruction on how to develop HPLC methods and how to troubleshoot HPLC problems. These courses are ideal for those who are using HPLC and want to improve their expertise. The cost for each one day course is  €275 + VAT per person. Contact us to find out about our academic discount and discounts for group bookings.

How to Develop HPLC Methods
(Click here for more information)
25th October 2011
Learn how to select appropriate method conditions and perform suitable investigative experiments to obtain a set of method parameters which enables the desired separation for mixtures of analytes.

Excellent course, found it very informative and useful." – Angela Boag
"Very enjoyable & informative, Oona was extremely helpful.” –Keighley Campbell

How to Develop HPLC Methods for Challenging Separations 
(Click here for more information)
26th October 2011
Learn how to implement strategies to achieve satisfactory separation for ‘complex’ samples and use computer modelling to develop robust and fit for purpose HPLC methods.

I enjoyed the exercises as they made you think and apply what we just learnt.” – Julie Cooper
"Very good, learnt a lot. Thank you.” – Sally Housden

How to Troubleshoot HPLC 
(Click here for more information)
27th October 2011
Learn how to find solutions for problems encountered when running HPLC analysis by diagnosing symptoms and implementing appropriate preventative measures.

"Excellently presented and questions answered consistently and clearly. Problem solving sessions very useful for building up knowledge learnt on the day.” – Robert Hetterley
I am very pleased that I learned a lot of new things today and I am more confident in troubleshooting HPLC.” – Anna-Marie Corina Cristea

On-site Training
(Click here for more information)
We also offer all these courses (and others) as on-site training where we visit your company to deliver the training. This may include customisation to meet your unique requirements and ‘hands-on’ sessions in your laboratories. Contact us for more information.

Wednesday, 8 June 2011

Interpreting a HPLC analytical method

PEAK SOLUTIONS
A resource for chromatographers

The detail which is included in a HPLC analytical method will dictate how much interpretation is required by the analyst when following the method. For a very detailed method the analyst should be able to follow it easily but if the method does not include certain details the analyst will have to make some decisions about how to perform the analysis.

Local laboratory proceures
Routinely parts of the HPLC analytical method are covered by the local procedures in the analytical laboratory and thus will not be included in the HPLC analytical method. Examples are:
The sequence of the injections to be performed.
The procedure to use when preparing the mobile phase, e.g. the grades of solvents which should be used.
The routine use of guard columns or cartridges.
The implementation of wash programmes for column post analysis.

A good knowledge of the local laboratory procedures is required to ensure that all requirements are met.

HPLC column not defined or unavailable
When following a pharmacopoeia method, the column to use will be defined by the bonded phase type (e.g. octadecyl silane) and the particle size range (e.g. 1.5 to 10µm). This makes it difficult to choose which column to use since these parameters may be used to describe hundreds of columns. It is up to the analyst to select a suitable column. There may be a preference in the laboratory for a particular column to use in these situations. Some method development may be necessary to achieve the correct results following the monograph.

Another problem related to columns which may be encountered is that the column needed for a method is no longer available. In this case an equivalent column needs to be sourced. To find an equivalent column a column classification system is required. There are a number of researchers working in this area and their work has produced data for comparing columns [1,2].

Dwell volume
When using gradient methods the effects of dwell volume can lead to changes in the retention times when methods are transferred between different instruments. Ideally methods are developed to be robust to changes in dwell volume but if the change results in reduced resolution a solution will be required.If the retention times are shorter than expected due to the difference in dwell volume then the current system has a smaller dwell volume than the previous system. A solution is to introduce a ‘hold’ of the initial gradient mobile phase composition at the beginning of the analysis. The length of the hold can be determined by measuring the dwell volume on the two systems and dividing the difference by the flow rate, or, it can be determined experimentally by injecting some test samples with different hold values programmed into the gradient table.If the retention times are longer than expected due to the difference in dwell volume then the current system has a larger dwell volume than the previous system. Unless the injector has a function whereby the injection can be performed after the gradient has started (unlikely in older systems which are more likely to have a large dwell volume) the method cannot be satisfactorily run on the instrument.

The dwell volume of an HPLC system is easy to measure:
  1. Remove the column from the system and use a short length of 0.010″ tubing to connect the injector directly to the detector.
  2. For solvent A, use HPLC grade water; for solvent B, add about 0.1% acetone to water (methanol or acetonitrile can be used instead of water).
  3. Set the detector wavelength to 265nm.
  4. Run a typical gradient from 0 to 100% B (e.g. 0-100% in 10 minutes at 1 mL/min flow).
  5. Record the detector signal during this gradient.
  6. Print out or display the "chromatogram" from the gradient run. It should look like Figure 1. Draw the best straight line fit to the flat portion at the beginning of the plot. Draw the best straight line fit to the linear ramp of the gradient. The time at which these two lines intersect is the dwell time (tD). The dwell volume is the product of the dwell time and the flow rate used for the test.
Figure 1


References:
1. D. Visky, E. Haghedooren, P. Dehouck, Zs. Kovács, K. Kóczián, B. Noszál, J. Hoogmartens, E. Adams, , J. Chromatogr. A, 1101, 103-114, 2006, ‘Facilitated column selection in pharmaceutical analyses using a simple column classification system’.(This research group provide their column classification data on this website: www.pharm.kuleuven.be/pharmchem.)
2. M.R. Euerby, P. Petersson, J. Chromatogr. A, 994, 13-36, 2003, ‘Chromatographic classification and comparison of commercially available reversed-phase liquid chromatographic columns using principal component analysis’.This research group has a collaboration with Advanced Chemistry Development (ACD/Labs) to provide a column selection tool (http://www.acdlabs.com/).

This blog post is an excerpt from 'An Introduction to HPLC for Pharmaceutical Analysis' by Oona McPolin, available to purchase through the MTS website.

Friday, 20 May 2011

Help on: Gradient programs for HPLC

MTS HELPDESK

Do you have any problems relating to analytical chemistry for pharmaceuticals or training? Send your questions to the MTS helpdesk using our contact form.

Question:
“How do you calculate the gradient program in HPLC method development?”

Answer:
“The aim of an HPLC method is to enable the separation of a mixture of components. This may be achieved by selecting a suitable mobile phase composition for a particular column which results in a peak for each component that is separated from other peaks, and is retained at a suitable retention time. The composition of the mobile phase may be isocratic, where it is held constant throughout each injection, or gradient, where the amount of the stronger solvent is increased throughout each injection. Whether you use isocratic or gradient conditions depends on the nature of your mixture of analytes. For example in reversed phase HPLC a mixture of analytes which have widely differing hydrophobicity is likely to require an unfeasibly long run time under isocratic conditions and will need to be analysed under gradient conditions.

For RP-HPLC, I suggest that you run your test sample using a full range gradient, e.g. 5 to 95 %B. An inspection of the resulting chromatogram will indicate whether the method is suitable for isocratic analysis (if the difference between the first and last peak is less than 25% of the gradient time) and if not, provides a starting point for gradient method development. Changing the gradient time, tG will make the gradient more or less steep, corresponding to stronger and weaker mobile phase composition in isocratic analysis.

For a detailed discussion on gradient method development you may wish to attend one of my training courses. ‘How to Develop HPLC Methods’ will be held next week in London and Milton Keynes. Future events will be publicised on this blog.”

Thursday, 19 May 2011

How to Develop HPLC Methods

This HPLC method development training course, sponsored by Phenomenex, was held on Tuesday and Wednesday of this week, in Crewe and Edinburgh respectively. The aim of the training was to describe a strategy which can be put in place to develop a new method quickly and easily. The feedback from the delegates was very positive. Comments included:
“Excellent course, found it very informative and useful."
"Very enjoyable & informative, Oona was extremely helpful.”
“I enjoyed the exercises as they made you think and apply what we just learnt.”

There are two more dates for this course next week, in London on Tuesday and in Milton Keynes on Wednesday. If you are interested in attending, contact Phenomenex (course code SS0-5947) to check if there are any places left. Tel: UK: 01625 501367 or email: ukinfo@phenomenex.com

Course Details:
Summary
Learn how to select appropriate method conditions and perform suitable investigative experiments to obtain a set of method parameters which enables the desired separation for mixtures of analytes.
This course is ideal for those who have experience of running HPLC methods and now want to learn how to develop new methods.

Course Outline
Developing an HPLC method using a 5-step strategy:
 Step 1: Setting suitable objectives for method development
 Step 2: Assessing all available information
 Step 3: Selecting suitable samples
 Step 4: Performing scouting experiments to select suitable initial conditions
 Step 5: Optimising the method to define method parameters which achieve the desired separation
This course focuses on reversed phase mode separations.

Practical Skills Acquired
This course will enable you to take a strategic approach to developing HPLC methods with an understanding of the factors which can be adjusted to manipulate the retention time of analytes. In addition you will be able to:
1. Define the objectives for the development of a HPLC analytical method.
2. Effectively assess all the available relevant information relating to the desired method, e.g. pKa of the analyte.
3. Select and prepare a suitable sample or samples to be used for the method development.
4. Select suitable scouting conditions to find a suitable column and mobile phase system.
5. Optimise the chromatographic conditions to result in the best possible separation.

Course price is £195 + VAT per delegate. The price includes: Full day training (including post training assessment), course literature, technical brochures, lunch and refreshments.

A follow up course: ‘How to Develop HPLC Methods for Challenging Separations’ (course code SS0-5946) is planned for September, at Crewe on the 13th and Milton Keynes on the 20th. The aim of this course is to enable development of methods for problem samples and analytes, examples being very polar molecules and samples containing numerous analytes.

Tuesday, 3 May 2011

Help on: Forced Degradation Studies

MTS HELPDESK
Do you have any problems relating to analytical chemistry for pharmaceuticals or training? Send your questions to the MTS helpdesk using our contact form.
Question:
What is meant by forced degradation studies?
Answer:
In a forced degradation study a molecule is subjected to extremes of heat, humidity, pH, light, etc. so that the molecule degrades. This study is commonly performed for pharmaceutical active pharmaceutical ingredients and formulations and is typically done for one of two reasons:

1. It provides information on the possible degradation pathways of the molecule and thus an assessment of its stability under different types of conditions. The information obtained is usually included in the marketing dossier for the drug.

2. It provides samples which can be used to develop a stability indicating method, i.e. a method which can detect and quantify degradation products. This method is used to assess shelf life during stability studies.

A problem with forced degradation studies is that the extreme conditions used may not be representative of the degradation under ‘normal’ conditions. For this reason the aim is to degrade the molecule of interest by no more than 10%.

Find out more about forced degradation studies on the MTS course, 'How to Develop Stability Indicating HPLC Methods', click here for more information.


 

Monday, 25 April 2011

System Suitability Failures

The purpose of system suitability testing is summarised by the USP as follows: "System suitability tests are an integral part of gas and liquid chromatographic methods. They are used to verify that the chromatographic system is adequate for the intended analysis. The tests are based on the concept that the equipment, electronics, analytical operations and samples to be analyzed constitute an integral system that can be evaluated as such."[1] In practice the testing consists of measurements performed on the chromatograms obtained for particular injections during the analysis which provide an indication of whether the HPLC method and system is performing as would be expected. Typical system suitability parameters, as defined by the FDA[2], are summarised in Table 1, with definition of terms for the parameters provided in Figure 1. Acceptance criteria based on recommendations provided by the FDA[2] are provided in Table 2, these are often implemented as a ‘generic’ set of conditions when new methods are developed. Additional requirements may be added as required, e.g. a measure of detector sensitivity may be assessed for a low level impurity method.


Table 1



Figure 1


Table 2

When a system suitability test fails, an investigation into the cause is required. Data generated from the analysis cannot be used. Ideally analytical results should not be generated until the system suitability test results have been shown to pass. In practice however, the processing for an HPLC analysis is usually automated and thus the results are often generated before the results of the system suitability test have been evaluated. In this situation it is very important that the documentation accompanying the analysis is very clear about why the results have been rejected. The available regulatory guidance[3,4] for the investigation of out of specification (OOS) and out of trend (OOT) results involves inspection of the system suitability conditions for the analysis, even though it should already have been assessed prior to accepting the analysis as valid. This may seem redundant but it is important to review all available data during this type of laboratory investigation. It is also possible that the system suitability testing for the method is inadequate and may need to be reviewed and updated.

It is desirable to minimise the occurrence of system suitability failures since each failure represents wasted time spent on analysis and additional time spent conducting an investigation into the failure. The consequences of the failure are not severe: the cause is documented and the analysis repeated. However, regular failures are of concern and may indicate a more serious root cause. The test measures the operation of an HPLC method in a holistic fashion and the reasons for failure of the test are varied. The most common reasons are related to:
1. Degradation of the HPLC column,
2. Competence of the analyst, and
3. Maintenance of the HPLC system.

Degradation of the HPLC column
The column has been singled out since it is a consumable item and it is expected that over time it will degrade and in the process have an effect on the chromatographic performance of the method. Resolution and efficiency are likely to decrease and tailing to increase over time. Under ideal conditions the primary purpose of the system suitability test would be to indicate when the column was no longer performing adequately for the method and should be replaced. In many laboratories the expected lifetime of a HPLC column for a particular method is not investigated and thus there is a risk that using a generic set of acceptance criteria for system suitability testing may result in a failure of the test when in fact the column is still performing adequately, or potentially (though less likely) a pass when the column has actually degraded past the point where acceptable results can be obtained.

Typically the number of injections that can be obtained for a column depends on the combination of the mobile phase, the system conditions, and the sample being analysed. Columns which are subjected to method conditions which include inorganic buffers, high pH or high temperature may result in a reduced number of injections, as will columns which are used for ‘dirty’ samples. An evaluation of the aging of the column for a method is advisable to set appropriate system suitability criteria. This may be carried out as a dedicated study during method development, or a column may be monitored in normal use of the method. Either way the data gained will provide a sound scientific basis for a set of relevant system suitability criteria. In addition, warning limits may be set which alert the operator that the column needs to be replaced soon, thus eliminating any system suitability failure due to the aging column.

Competence of the analyst
The analyst performing HPLC analysis must not only be competent in the preparation of mobile phase and test solutions but also in setting up the HPLC system. Errors in mobile phase preparation can affect the capacity factor and thus the retention time of analytes. If the analyst introduces contamination in the mobile phase this may introduce extra (unwanted) peaks, decrease resolution and degrade the overall chromatography. Errors in preparation of test solutions can affect the accuracy and precision of the analytical results. Examples of problems due to incorrect set up of the HPLC system include unsteady and noisy baselines if the pump is not adequately primed, peak broadening effects (decreased efficiency) due to unsatisfactory connections and drifting baselines and retention times if the temperature is not controlled.

If the operator has a good understanding of the method, this will help to ensure that important method parameters are controlled, e.g. in some HPLC methods the retention times of the analytes are very sensitive to the pH of the buffer in the mobile phase and thus strict control of the pH is critical. If the analyst is aware of this requirement they will be more careful during the pH adjustment task. Suitable training is essential to ensure that system suitability failures are not due to the competence of the analyst.

Maintenance of the HPLC System
If not correctly maintained the HPLC instrumentation can contribute to system suitability testing failures, e.g. worn injector parts may result in repeatability failure, an aged lamp in a UV detector can cause baseline noise and have a detrimental effect on the quantification of low level analytes. The system suitability test is not a substitute for the performance qualification of the HPLC instrument since it is method based rather than instrument based. Although it provides assurance that the method may be used satisfactorily at that time it does not test that the instrument is functioning correctly, e.g. accuracy of flow rate, wavelength, injection volume, temperature, etc. Regular maintenance and performance verification should be scheduled to ensure that the HPLC system does not contribute to system suitability testing failures.

In summary, system suitability testing failures can be reduced by a combination of three measures:

1. Set system suitability criteria which relate specifically to the method in use. A column degradation study will identify the parameters of resolution, tailing and efficiency which indicate that a new column should be used.

2. Ensure that HPLC operators have received suitable training and are familiar with the method in use, and in particular, the robustness issues relating to the method.

3. Implement a regular maintenance and performance verification procedure for HPLC systems.

References:
1. United States Pharmacopeia (USP), Chromatography <621>
2. Reviewer Guidance: Validation of Chromatographic Methods, US Food and Drug Administration, 1994
3. Guidance for Industry: Investigating Out-of-Specification (OOS) Test Results fror Pharmaceutical Production US Food and Drug Administration, 2006
4. Out of Specification Investigations, MHRA

Monday, 11 April 2011

MTS Recommends... Measuring Volume - Beakers, Cylinders, Erlenmeyer Flasks, & Volumetric Flasks


This free video is a useful learning resource which describes the different types of volumetric glassware and how to use them.

Thursday, 31 March 2011

MTS Recommends... Chiral Method Development Screening Techniques

Chiral Method Development Screening Techniques: A practical guide and new approaches in LC-MS by David S Bell & Denise Wallworth, Chromatography Today, September 2009

When faced with developing a chiral HPLC method it is difficult to predict which type of chiral column will give the best results for your analyte. Screening a range of columns and conditions allows you to pick the most suitable. This article describes a screening approach for chiral HPLC.

Monday, 21 March 2011

Phenomenex Press Release for HPLC Training Courses in UK

The MTS training consultant, Oona McPolin, will be delivering a series of HPLC training courses in partnership with Phenomenex over 2011. The following press release about the courses has been released by Phenomenex:

These new training courses are designed to cater for novice chromatographers through to experienced method developers. This new series of courses will provide you with practical skills and knowledge to utilise immediately in your own working environment.
At locations across the UK: London, Milton Keynes, Crewe, Glasgow & Edinburgh

Courses being run in 2011 are:

How to….
Course 1: Run HPLC methods.
Course 2: Develop HPLC methods
Course 3: Validate chromatographic methods
Course 4: Develop HPLC methods for challenging separations
Course 5: Troubleshoot HPLC

All courses cost just £195+ VAT (this includes full course materials, refreshments, lunch and post course assessment with certificate of completion).

Download Course Brochure: Click Here

Although sponsored by Phenomenex, these courses are written and presented by Mrs Oona McPolin (BSC, MSc, CSci, CChem, MRSC) who has considerable experience and is fully qualified in the areas of both pharmaceutical analysis and training practice. She worked as an analytical chemist for over 10 years at a major global pharmaceutical company, on a range of drug development projects also with responsibility for many pharmaceutical analysis training programmes. Mrs McPolin has obtained the industry standard qualification for training, the Certificate in Training Practice, from the Chartered Institute of Personnel and Development (CIPD).

For more information or to book a place, please contact Phenomenex Today.
Tel: 01625 501367(UK) 01 247 5405(Eire) Email: ukinfo@phenomenex.com

Friday, 18 March 2011

Troubleshooting Tip: Baseline noise

PEAK SOLUTIONS 
A resource for chromatographers

If there is regular noise in your HPLC baseline then you need to figure out what is causing it before you can find a solution. A good first step is to turn off the pump and monitor the output from your detector. If the noise is still present then the problem is not linked to the flow of mobile phase. For a UV detector the most likely source of the noise is the lamp, either it has aged to a point where it needs replaced, or it is defective. Replacing the lamp should resolve the problem. If the noise stops when the pump is stopped then it is linked to the flow of mobile phase. Likely causes are:
  • Trapped air in the flow cell - try flushing with degassed mobile phase, or a strong solvent
  • A leak - carefully check your system for leaks and refit or replace the affected part
  • Temperature fluctuations - control the temperature
  • Incomplete mobile phase mixing - if you are using the HPLC system to mix the mobile phase, try mixing the mobile phase by hand and see if the noise goes away. 
  

Tuesday, 15 March 2011

MTS Recommends... LC Modelling using DryLab

3-Dimensional Retention Modelling of Gradient Time, Ternary Solvent-Strength and Temperature of the Reversed-phase Gradient Liquid Chromatography of a Complex Mixture of 22 Basic and Neutral Analytes using DryLab® 2010 by Melvin R Euerby, Gesa Schad, Hans-Jürgen Rieger & Imre Molnár, Chromatography Today, December 2010

In this article the HPLC modelling software, DryLab, is used to model a separation of pharmaceutical analytes using gradient time, temperature and ternary composition. Probably the most common approach for computer modelling is using a combination of gradient time and temperature, so this approach adds the potential use of ternary mixtures of mobile phases and brings the increased selectivity options that this will provide. This article will be of interest to anyone using computer simulation as part of their method development strategy.

Friday, 11 March 2011

HPLC Training in Belfast

Last Tuesday, the MTS training consultant, Oona McPolin, delivered the training course ‘How to Troubleshoot HPLC’ at the Culloden Hotel in Belfast. The training was well received generating very positive feedback. This one day course is designed with two aims, to provide: 1 - a full understanding of how HPLC systems work so that users can prevent problems from occurring, and 2 - knowledge of the symptoms of common problems to allow successful troubleshooting. Thank you to everyone who took part and don’t forget to get in touch if you have any HPLC troubleshooting questions.

We plan to organise further training sessions later this year on the topics of HPLC method development and method validation at venues in Ireland. Get in touch if you are interested in attending these courses.

Monday, 28 February 2011

Help on: ‘Dry’ solvents for HPLC?

MTS HELPDESK
Do you have any problems relating to analytical chemistry for pharmaceuticals or training? Send your questions to the MTS helpdesk using our contact form.

Question:
"I have observed that in one of your answers for a previous helpdesk blog, ‘Retention time shifts in NP-HPLC’, you used the term ‘dry’ solvents. What is the difference between dry solvents and ordinary HPLC solvents, and what is the use of anhydrous sodium sulfite in mobile phase?"

Answer:
"Anhydrous or dry solvents have been treated to remove trace amounts of water that may be present. It is possible to buy anhydrous solvents from your normal solvent supplier or you can use a variety of methods to dry solvents in your laboratory. The most suitable method depends on the actual solvent in question but typical methods include addition of anhydrous salts, distillation and use of a molecular sieve (usually 0.4nm). The suggested use of solvents which are half saturated with water (in the previous blog post) is a method by which retention times can be kept reasonably constant by controlling the amount of water present in the mobile phase, since the amount of residual water present in mobile phase can affect retention times considerably.

Another source of trace amounts of water is the sample which is injected onto your HPLC column, thus the suggestion to consider drying this sample by the addition of anhydrous sodium sulphate. This salt is widely used as an inert drying agent for removing traces of water from organic solutions in the laboratory. I would not recommend adding it to the mobile phase. The aim in NP-HPLC is to achieve a fairly consistent amount of water in the mobile phase rather than using completely dry solvents. Once opened dry solvents will absorb water over time and this change will affect retention times in NP-HPLC."

Wednesday, 23 February 2011

Tuesday, 1 February 2011

HPLC Troubleshooting Training Course in Belfast

Does HPLC ever leave you feeling like this?
You need our training course How To Troubleshoot HPLC.

Learn how to find solutions for problems encountered when running HPLC analysis by diagnosing symptoms and implementing appropriate preventative measures. This course is ideal for those who have experience of using HPLC and now want to develop their skills further.

New date now available:
8th March 2011
The Culloden Hotel, Belfast, Northern Ireland.

Book online or contact us for more information

Just £195 + VAT per person if booked before 22nd February 2011. Discounts for groups are available. This includes: Comprehensive handouts; access to online training resources; post training assessment and certificate of training; lunch and refreshments.
This course is eligible for Refer Your Friends.

Let us know if you would like this training course at a location near you.

Friday, 28 January 2011

MTS Website Updates

We have made some updates to the MTS website. There is now a Course List so that you can easily view the range of courses that are available. In addition to a brief course summary and information on costs, the delivery options for each course are listed so that you can see at a glance whether the course is available by eLearning, on-site, at an external location, etc. The Resources section has also been updated and all past issues of Analyse This are now available on a dedicated webpage. We also have a webpage for our new Refer Your Friends scheme.

Thursday, 20 January 2011

MTS Recommends... Using Temperature in LC Method Development

The Use of Temperature for Method Development in LC by Gerd Vanhoenacker , Frank David & Pat Sandra, Chromatography Today, September 2010

Since the ionisation equilibria of analytes and mobile phase for polar and ionisable analytes is temperature dependent, temperature can be used as a useful selectivity parameter when developing methods for these types of analytes. This technical note discusses the range of temperatures which can be used for method development and the different types of available LC columns which can be used at high temperatures.

Tuesday, 18 January 2011

Help on: Equilibration of HPLC columns between gradient method injections

MTS HELPDESK
Do you have any problems relating to analytical chemistry for pharmaceuticals or training? Send your questions to the MTS helpdesk using our contact form.

Question:
“I have a question about column re-equilibration after each injection ready for the next one. Firstly, is there a way how to calculate how long the re-equilibration step should be (or is it more of experience and trial and error)? I am using a 30 x 4.6mm column (2.7 particle size), HP1100 pump, 1.7ml/min flow rate, and 2 min gradient method (as you can see I’m after a fast turn-around!). I’m just a bit worried if I’m giving the column enough time to re-equilibrate ready for the following injection. My gradient is as follows:

Time - %A - %B
0.00 - 90.0 - 10.0
0.10 - 90.0 - 10.0
0.50 - 10.0 - 90.0
1.50 - 10.0 - 90.0
1.60 - 90.0 - 10.0
2.00 - 90.0 - 10.0

I am also taking advantage of the next injection’s injection time (25 seconds) which I’m assuming can also be used as part of the re-equilibration step? I have done a series of injections (~10) and the retention times are pretty constant using this 2 min method. Though I was also wondering over how many injections could you justify the method reliable, would 10 be enough?”

Answer:
“A general rule for equilibration is to allow 10 column volumes of mobile phase to pass through the column. Column volume refers to the amount of space in a column which can be taken up by the mobile phase, or the space not taken up by stationary phase packing. A previous blog on column equilibration describes the calculations for determination of this value and the MTS HPLC calculator can convert this to a time value, you may find these helpful.

For your method 10 column volumes is equal to 2.1 minutes. Given the length of your analysis I think that this would be too much time to spend on re-equilibration. Your current equilibration time is half a minute in the gradient table and another 25 sec for the injection which equates to about 5 column volumes. I feel that this should be sufficient if the method can be demonstrated to be working reproducibly. The signs of insufficient equilibration are baseline problems and variation in retention time so if you can show that these are constant then you can justify re-equilibration using 5 column volumes.

The method conditions will determine whether 5 column volumes is enough for re-equilibration, i.e. how different the starting and end conditions of the gradient are. Although your gradient is quite steep which means it has to go back from 90% B to 10% B in the re-equilibration, the 10 injections that you have performed give a good indication that the method is working fine. It would probably be good to back this up with some results over a longer time frame. Rather than setting up an experiment to test this I would suggest monitoring the use of the method when you analyse samples.”

Tuesday, 4 January 2011

HPLC Training Video Wins Silver Award in Tutorial Competition

The training video ‘A Brief Guide to HPLC Instruments’ has won a Silver Award in the Year of Education in Separation Science Tutorial Competition. Click here to read more and view the three winning entries.