Method Development in UHPLC

Method Development in UHPLC

The rules for developing a new method in UHPLC are slightly different from those of conventional HPLC because it is necessary to account for the backpressure constraint generated by the use of small particles.

3.1. Choice of column dimensions 

Depending on the supplier, it is possible to find some columns dedicated to UHPLC with internal diameters of 1, 2.1 and 4.6 mm. As discussed previously, the 4.6 mm I.D. column is not of great interest because of the significant frictional heating generated by high mobile phase flow rates, generating a lack of repeatability of retention times and some potential difficulties in transferring methods from conventional HPLC. In addition, the consumption of organic solvent is critical as the flow rate should be in the range 3-5 mL/min. Concerning the 1mm I.D column, the effect of frictional heating should be neglected even at 1000 bar but the compatibility between this column geometry and any UHPLC instrument is extremely critical (particularly for tubing geometry). Due to these statements, the 2.1 mm I.D. column should be considered as optimal for UHPLC operation.

Regarding the column length, it should be selected according to the required efficiency (in isocratic mode) or peak capacity (in gradient mode). It has to be noted that there is no real limitation in UHPLC column length. Numerous suppliers propose some 150mm columns which can be coupled in series using low volume tubing if an experiment has to be performed with very long columns.

In the isocratic mode, it is well known from the basic equations of chromatography that efficiency is directly proportional to the column length. Therefore, a 50x2.1 mm, 1.9 µm column should generate around 10,000 plates, while the efficiency is increased to 20,000 and 30,000 plates with a 100 and 150x2.1 mm, 1.9 µm column respectively. Therefore, the column length should be chosen according to the requirement of the separation and the longest column always provides the highest efficiency. However, with 150 mm or longer columns, the mobile phase flow rate should be adapted to avoid over-pressurizing the analytical system.

In the gradient mode, the relationship between column length and chromatographic performance (expressed as peak capacity in gradient mode) is not trivial.  In fact, the peak capacity depends both on efficiency and column dead time, but each to a different extent. Therefore,  the column length should be adapted according to the gradient time  and the longest column doesn’t necessarily provide  the highest peak capacity. It can be demonstrated that a 50x2.1 mm, 1.9 µm column has to be selected for gradient times lower than 5 minutes. The 100x2.1 mm, 1.9 µm column gives optimal performance for gradient times between 5 and 20 minutes and finally, the 150x2.1 mm, 1.9 µm column should only be used with gradients longer than 20 minutes.

3.2. Choice of mobile phase conditions

In UHPLC, the mobile phase flow rate has to be selected according to the Van Deemter curve (similarly to conventional HPLC) but also, according to the backpressure generated. For compounds with molecular weights in the range 100-400 g.mol^-1 , the optimal flow rate in isocratic mode for a 2.1 mm I.D. column packed with 1.9 µm particles is around 400-600 µL/min. Due to the low mass transfer resistance generated by small particles (because of the reduced diffusion path), it is even possible to work up to 1000 µL/min, with a limited loss in efficiency (around 20%). When dealing with larger molecules, the mobile phase flow rate should be reduced to 200-400 µL/min due to a reduction of diffusion coefficients.

The rules in gradient mode are different, where the highest flow rate always provides the best peak capacity because it is dependent on the column dead time and, to a lesser extent, on efficiency. Therefore, the flow rate for gradient UHPLC experiments should be elevated but at the maximum equal to 80-90% of the maximum pressure withstood by the instrument. This solution is recommended to attain a sufficient level of robustness and to handle unexpected changes in column backpressure after hundreds of injections.

Regarding the mobile phase temperature, it could be valuable to work in UHPLC at  a mobile phase temperature of 40-50°C  instead of room temperature. With this strategy, the mobile phase viscosity is reduced and the backpressure diminishes by about 30% (at 50°C for an acetonitrile-water mobile phase) without affecting chromatographic performance.

Finally, it is well known that the viscosity of acetonitrile-water hydro-organic mixtures is on average 1.5 to 2-fold lower than that of methanol-water. Therefore, in the case of method development, the initial choice for mobile phase is acetonitrile as it generates significantly lower backpressure and more possibilities in UHPLC

compared to methanol (particularly for the choice of column length).

3.3. Decision tree for method development

On the basis of the above discussion, it is possible to establish some generic conditions for the UHPLC method development: the column should be initially a C18 with geometry of 50x2.1 mm, 1.9 µm operating at a temperature of 40-50°C. The mobile phase should c onsist in a mixture of acetonitrile and buffer. It is generally better to begin the experiments in the gradient mode at a mobile phase flow rate close to the maximal pressure accepted by the UHPLC instrument. Regarding the choice of gradient time, it is extremely different in UHPLC compared to regular HPLC. Therefore, table 1 summarizes the optimal gradient time for various sets of UHPLC conditions.

If the selectivity with the generic conditions previously described is not acceptable, it is possible to adapt various parameters (e.g. mobile phase pH, nature of the stationary phase and organic modifier). In UHPLC, the first parameter to change is the mobile phase pH, then the column chemistry and finally the organic modifier nature (because of the constraint in pressure with UHPLC).

HPLC: Analytical Method Vallidation

Method validation. Proper validation of analytical methods is important for pharmaceutical analysis when ensurance of the continuing efficacy and safety of each batch manufactured relies solely on the determination of quality. The ability to control this quality is dependent upon the ability of the analytical methods, as applied under well-defined conditions and at an established level of sensitivity, to give a reliable demonstration of all deviation from target criteria. Analytical method validation is now required by regulatory authorities for marketing authorizations and guidelines have been published. It is important to isolate analytical method validation from the selection and development of the method. Method selection is the first step in establishing an analytical method and consideration must be given to what is to be measured, and with what accuracy and precision. 
Method development and validation can be simultaneous, but they are two different processes, both downstream of method selection. Analytical methods used in quality control should ensure an acceptable degree of confidence that results of the analyses of raw materials, excipients, intermediates, bulk products or finished products are viable. Before a test procedure is validated, the criteria to be used must be determined. 
Analytical methods should be used within good manufacturing practice (GMP) and good laboratory practice (GLP) environments, and must be developed using the protocols set out in the International Conference on Harmonization (ICH) guidelines (Q2A and Q2B).^1,2 The US Food and Drug Administration (FDA)^3,4 and US Pharmacopoeia (USP)⁵ both refer to ICH guidelines. The most widely applied validation characteristics are accuracy, precision (repeatability and intermediate precision), specificity, detection limit, quantitation limit, linearity, range, robustness and stability of analytical solutions. Method validation must have a written and approved protocol prior to use.⁶

^{This article reviews and demonstrates practical approaches to analytical method validation with reference to an HPLC assay of progesterone (Figure 2) in a gel formulation. Progesterone is widely used for dysfunctional uterine bleeding or amenorrhoea,7,8 for contraception (either alone or with, for example, oestradiol or mestranol in oral contraceptives) and in combination with oestrogens for hormone replacement therapy in postmenopausal women.9,10Experimental Chemicals and reagents All chemicals and reagents were of the highest purity. HPLC-grade methanol was obtained from Merck (Darmstadt, Germany). Progesterone reference standard was purchased from Sigma Chemicals (St Louis, Missouri, USA). Deionized distilled water was used throughout the experiments.}

Equation 1 and Figure 4: HPLC chromatograms of (a) progesterone reference standard; (b) separation of progesterone gel sample; (c) placebo formulation

HPLC instrumentation The HPLC systems used for the validation studies consisted of Series 200 UV/Visible Detector, Series 200 LC Pump, Series 200 Autosampler and Series 200 Peltier LC Column Oven (all Perkin Elmer, Boston, Massachusetts, USA). The data were acquired via TotalChrom Workstation (Version 6.2.0) data acquisition software (Perkin Elmer), using Nelson Series 600 LINK interfaces (Perkin Elmer). 
All chromatographic experiments were performed in the isocratic mode. The mobile phase was a methanol/water solution (75:25 v/v). The flow rate was 1.5 mL/min and the oven temperature was 40 ºC. The injection volume was 20 μL and the detection wavelength was set at 254 nm. The chromatographic separation was on a 25034.6 mm ID, 10 μm C₁₈ μ-Bondapak column (Waters, Milford, Massachusetts, USA). 

Results and discussionLinearity and range The linearity of a test procedure is its ability (within a given range) to produce results that are directly proportional to the concentration of analyte in the sample. The range is the interval between the upper and lower levels of the analyte that have been determined with precision, accuracy and linearity using the method as written. ICH guidelines specify a minimum of five concentration levels, along with certain minimum specified ranges. For assay, the minimum specified range is 80–120% of the theoretical content of active. Acceptability of linearity data is often judged by examining the correlation coefficient and y-intercept of the linear regression line for the response versus concentration plot. The regression coefficient (r²) is .0.998 and is generally considered as evidence of acceptable fit of the data (Figure 3) to the regression line. The per cent relative standard deviation (RSD), intercept and slope should be calculated. In the present study, linearity was studied in the concentration range 0.025–0.15 mg/mL (25–150% of the theoretical concentration in the test preparation, n=3) and the following regression equation was found by plotting the peak area (y) versus the progesterone concentration (x) expressed in mg/mL: y53007.2x14250.1 (r²51.000). The demonstration coefficient (r²) obtained for the regression line demonstrates the excellent relationship between peak area and concentration of progesterone. The analyte response is linear across 80-120% of the target progesterone concentration. 

Table V: Demonstration of the repeatability of the HPLC assay for progesterone.

Accuracy A method is said to be accurate if it gives the correct numerical answer for the analyte. The method should be able to determine whether the material in question conforms to its specification (for example, it should be able to supply the exact amount of substance present). However, the exact amount present is unknown, which is why a test method is used to estimate the accuracy. Furthermore, it is rare that the results of several replicate tests all give the same answer, so the mean or average value is taken as the estimate of the accurate answer. Some analysts adopt a more practical attitude to accuracy, which is expressed in terms of error. The absolute error is the difference between the observed and the expected concentrations of the analyte. Percentage accuracy can be defined in terms of the percentage difference between the expected and the observed concentrations (Equation 1). 
Percentage accuracy tends to be lower at the lower end of the calibration curve. The term accuracy is usually applied to quantitative methods but it may also be applied to methods such as limit tests. Accuracy is usually determined by measuring a known amount of standard material under a variety of conditions but preferably in the formulation, bulk material or intermediate product to ensure that other components do not interfere with the analytical method. For assay methods, spiked samples are prepared in triplicate at three levels across a range of 50-150% of the target concentration. The per cent recovery should then be calculated. The accuracy criterion for an assay method is that the mean recovery will be 100±2% at each concentration across the range of 80-120% of the target concentration. To document accuracy, ICH guidelines regarding methodology recommend collecting data from a minimum of nine determinations across a minimum of three concentration levels covering the specified range (for example, three concentrations, three replicates each). 
In the present study, the accuracy of the method was evaluated by recovery assay, adding known amounts of progesterone reference standard to a known amount of gel formulation, to obtain three different levels (50, 100 and 150%) of addition. The samples were analysed, and mean recovery and %RSDs calculated. The data presented in Table IV show that the recovery of progesterone in spiked samples met the evaluation criterion for accuracy (100±2.0% across 80–120% of target concentrations). 
Specificity Developing a separation method for HPLC involves demonstrating specificity, which is the ability of the method to accurately measure the analyte response in the presence of all potential sample components. The response of the analyte in test mixtures containing the analyte and all potential sample components (placebo formulation, synthesis intermediates, excipients, degradation products and process impurities) is compared with the response of a solution containing only the analyte. Other potential sample components are generated by exposing the analyte to stress conditions sufficient to degrade it to 80–90% purity. For bulk pharmaceuticals, stress conditions such as heat (50–60 ºC), light (600 FC of UV), acid (0.1 M HCl), base (0.1 M NaOH) and oxidant (3% H₂O₂) are typical. For formulated products, heat, light and humidity (70-80% RH) are often used. The resulting mixtures are then analysed, and the analyte peak is evaluated for peak purity and resolution from the nearest eluting peak. 

Table VI: Demonstration of the intermediate precision of the HPLC assay

Once acceptable resolution is obtained for the analyte and potential sample components, the chromatographic parameters, such as column type, mobile phase composition, flow rate and detection mode, are considered set. An example of specificity criterion for an assay method is that the analyte peak will have baseline chromatographic resolution of at least 2.0 from all other sample components. In this study, a weight of sample placebo equivalent to the amount present in a sample solution preparation was injected to demonstrate the absence of interference with progesterone elution (Figure 4). 
Precision Precision means that all measurements of an analyte should be very close together. All quantitative results should be of high precision - there should be no more than a ±2% variation in the assay system. A useful criterion is the relative standard deviation (RSD) or coefficient of variation (CV), which is an indication of the imprecision of the system (Equation 2). 
According to the ICH,² precision should be performed at two different levels - repeatability and intermediate precision. Repeatability is an indication of how easy it is for an operator in a laboratory to obtain the same result for the same batch of material using the same method at different times using the same equipment and reagents. It should be determined from a minimum of nine determinations covering the specified range of the procedure (for example, three levels, three repetitions each) or from a minimum of six determinations at 100% of the test or target concentration. 
Intermediate precision results from variations such as different days, analysts and equipment. In determining intermediate precision, experimental design should be employed so that the effects (if any) of the individual variables can be monitored. Precision criteria for an assay method are that the instrument precision and the intra-assay precision (RSD) will be ≤2%. 
In this study, the precision of the method (repeatability) was investigated by performing six determinations of the same batch of product. The resulting data are provided in Table V, which show that the repeatability precision obtained by one operator in one laboratory was 0.28% RSD for progesterone peak area and, therefore, meets the evaluation criterion

Table VII: Stability results of progesterone samples and standard solutions (n53).

The intermediate precision was demonstrated by two analysts, using two HPLC systems and who evaluated the relative per cent purity data across the two HPLC systems at three concentration levels (50%, 100%, 150%) that covered the assay method range (0.025–0.15 mg/mL). The mean and RSD across the systems and analysts were calculated from the individual relative per cent purity mean values at 50%, 100% and 150% of the test concentration. The data are presented in Table VI, and show ≤2.0% RSD, therefore, meeting the evaluation criterion. Limits of detection and quantitation The limit of detection (LOD) is defined as the lowest concentration of an analyte in a sample that can be detected, not quantified. It is expressed as a concentration at a specified signal:noise ratio,² usually 3:1. The limit of quantitation (LOQ) is defined as the lowest concentration of an analyte in a sample that can be determined with acceptable precision and accuracy under the stated operational conditions of the method. The ICH has recommended a signal:noise ratio 10:1. LOD and LOQ may also be calculated based on the standard deviation of the response (SD) and the slope of the calibration curve(s) at levels approximating the LOD according to the formulae: LOD53.3(SD/S) and LOQ510(SD/S). 
The standard deviation of the response can be determined based on the standard deviation of the blank, on the residual standard deviation of the regression line, or the standard deviation of y-intercepts of regression lines. The method used to determine LOD and LOQ should be documented and supported, and an appropriate number of samples should be analysed at the limit to validate the level. In this study, the LOD was determined to be 10 ng/mL with a signal:noise ratio of 2.9. The LOQ was 20 ng/mL with a signal:noise ratio of 10.2. The RSD for six injections of the LOQ solution was ≤2%. 
Analytical solution stability Validation of sample and standard solution preparation may be divided into sections, each of which can be validated. These include extraction; recovery efficiency; dilution process when appropriate; and addition of internal standards when appropriate. Although extraction processes do not actually affect the measuring stage they are of critical importance to the analytical test method as a whole. The extraction process must be able to recover the analyte from the product; it must not lose (for example, by oxidation or hydrolysis) any of the analyte in subsequent stages, and must produce extraction replicates with high precision. For example, during analysis of an ester prodrug the extraction process involves the use of strongly alkaline or acid solutions, it may cause some of the prodrug to be hydrolysed and, therefore, give false results. 
Reference substances should be prepared so that they do not lose any of their potency. Thus it is necessary to validate that the method will give reliable reference solutions that have not been deactivated by weighing so little that an error is produced; adsorption onto containers; decomposition by light; and decomposition by the solvent. If the reference is to be made up from a stock solution then it must be validated that the stock solution does not degrade during storage. Reagent preparation should be validated to ensure that the method is reliable and will not give rise to incorrect solutions, concentrations and pH values. 
Samples and standards should be tested during a period of at least 24 h (depending on intended use), and component quantitation should be determined by comparison with freshly prepared standards. For the assay method, the sample solutions, standard solutions and HPLC mobile phase should be stable for 24 h under defined storage conditions. Acceptable stability is ≤2% change in standard or sample response, relative to freshly prepared standards. The mobile phase is considered to have acceptable stability if aged mobile phase produces equivalent chromatography (capacity factors, resolution or tailing factor) and the assay results are within 2% of the value obtained with fresh mobile phase. 
In the present study, the stabilities of progesterone sample and standard solutions were investigated. Test solutions of progesterone were prepared and chromatographed initially and after 24 h. The stability of progesterone and the mobile phase were calculated by comparing area response and area per cent of two standards with time. Standard and sample solutions stored in a capped volumetric flask on a lab bench under normal lighting conditions for 24 h were shown to be stable with no significant change in progesterone concentration during this period (Table VII). 
Robustness Robustness measures the capacity of an analytical method to remain unaffected by small but deliberate variations in method parameters. It also provides some indication of the reliability of an analytical method during normal usage. Parameters that should be investigated are per cent organic content in the mobile phase or gradient ramp; pH of the mobile phase; buffer concentration; temperature; and injection volume. These parameters may be evaluated one factor at a time or simultaneously as part of a factorial experiment. The chromatography obtained for a sample containing representative impurities when using modified parameter(s) should be compared with the chromatography obtained using the target parameters. 
Conclusion Method development involves a series of sample steps; based on what is known about the sample, a column and detector are chosen; the sample is dissolved, extracted, purified and filtered as required; an eluent survey (isocratic or gradient) is run; the type of final separation (isocratic or gradient) is determined from the survey; preliminary conditions are determined for the final separation; retention efficiency and selectivity are optimized as required for the purpose of the separation (quantitative, qualitative or preparation); the method is validated using ICH guidelines. The validated method and data can then be documented. 
References 1. International Conference on Harmonization, "Q2A: Text on Validation of Analytical Procedures," Federal Register 60(40), 11260–11262 (1995). 
2. International Conference on Harmonization, "Q2B: Validation of Analytical Procedures: Methodology; Availability," Federal Register 62(96), 27463–27467 (1997). 
3. FDA, "Analytical Procedures and Methods Validation: Chemistry, Manufacturing and Controls Documentation; Availability," Federal Register (Notices) 65(169), 52776–52777 (2000). 
4. www.fda.gov/cder/guidance/cmc3.pdf

5. USP 25–NF 20, Validation of Compendial Methods Section (1225) (United States Pharmacopeal Convention, Rockville, Maryland, USA, 2002) p 2256. 
6. G.A. Shabir, "Validation of HPLC Chromatography Methods for Pharmaceutical Analysis. Understanding the Differences and Similarities Between Validation Requirements of FDA, the US Pharmacopeia and the ICH," J. Chromatogr. A. 987(1-2), 57-66 (2003). 
7. C.E. Wood, "Medicare Program; Changes to the Hospital Outpatient Prospective,"Med. J. Aust. 165, 510–514 (1996). 
8. A. Prentice, "Medical Management of Menorrhagia," Br. Med. J. 319, 1343–1345 (1999). 
9. D.T. Baired and A.F. Glasier, "Hormonal Contraception," New Engl. J. Med. 328, 1543–1549 (1993). 
10. P.E. Belchetz, "Hormonal Treatment of Postmenopausal Women," New Engl. J. Med. 330, 1062–1071(1994).

Optimizing the Separation : HPLC

Optimizing the Separation

Once you have a separation you may want to optimize it.  You may wish to shallow out the gradient to improve the separation, or you may wish to shorten the run time.  Taking the illustration above one can see that all of the peptides are out by 40 minutes.  This does not mean that we can change this 80 min run into a 40 min run, but there is room for improvement.  The first step in the optimization is to determine the %B at which the last peak elutes.  If you look at the blue gradient line you might guess that the last peak elutes near 40%B but this would be incorrect.  All HPLC systems have a gradient delay.  The gradient delay is the time between when the software tells the pumps to start pumping at a certain mobile phase composition and the time it takes for that solvent composition to reach the column and have an effect.  A good guess for a gradient delay is 10 minutes.  This would mean that our guess for the final mobile phase composition for the 40 min peak would be approximately 30%B.  To observe the gradient delay time look at the illustration above and observe that the baseline returns to the starting conditions at 70 minutes and not at 60.1 minutes when our pumps have gone back to 2% B.  One must take care to avoid having the last peak elute on the "equilibration cliff", (at 70 min. in this example).  This can be avoided by ending the gradient at a %B that is slightly higher than that required by the last component.
Based on the separation shown at the top of this section one could rewrite the gradient to look like this:

This would make the gradient shallower and possibly give a better peak separation.  To shorten the run time one could rewrite the gradient to look like this:

This last change would cut 30 min. from the analysis time.  Shorter analysis times are always better for work efficiency.  With every minute you can cut from the HPLC method without sacrificing your chromatographic goals you will be rewarded with better work efficiency. With this change the last peak would most likely still elute at 40 minutes and the peptide separation would essentially remain the same as in the initial 60/60 analysis.
What is HPLC Equilibration?

The column must be equilibrated, re-equilibrated to the initial high aqueous solvent composition before another analysis can be performed.  Normally this re-equilibration is stuck onto the end of the gradient.  How much equilibration time is enough?  As a rule of thumb we give 20 minutes.  In reality it depends on the column length, flow rate and the hydrophobicity of your peptides.  Some chromatographers use 10 minutes as their standard equilibration time.  Equilibration is all about fitness of purpose.  You should determine the the equilibration time experimentally, the criteria will be, does my analyte really stick to the column and chromatograph appropriately and reproducibly with subsequent analyses. If you choose to do this part of the method development you will undoubtedly be rewarded with improved chromatography and better cycle time.

Should I Control Column Temperature?
Yes.  Scientists are control freaks.  If you can control a variable, control it!  Actually if you are performing automated analyses over a long period of time peak retention times can drift with changing ambient temperature.  It is common for many companies and institutions shut down the air conditioning at night to save money, which could result in shifting peak retention times due to dramatic changes in ambient temperature.  Many HPLCs provide the option to control column compartment temperature.  If your HPLC does not have this capability a heated column jacket can be purchased from many suppliers.  The most common running temperature is 40°C, this places the column compartment well above even the most extreme ambient temperature fluctuations.  In addition to maintaining constant temperature, temperature can be used to influence the chromatographic separation.  No chromatographic study is complete without a temperature study.  In our experience higher temperature is better, peaks will be sharper and elute earlier. It is not too uncommon to perform chromatography at 60°C and some daredevils even go to 80°C.  Remember though that higher temperature will lead to a shorter column lifetime and some columns may not be able to tolerate 60°C.  Consult the manufactures recommendations when experimenting with high temperature. After your runs are complete for the day it is advised that you turn off your column heater since high temperature leads to stationary phase deterioration.

Preparing for the First Run of the Day
One observation is that if you start up a reverse phase analysis from a dead stop with a column that has perhaps been sitting in high aqueous conditions for up to 10 hours the analysis will give irreproducible results.  Conventional wisdom has it, you want to first flush the column with the highest % organic of your method for at least 3 column volumes and then bring it back to the equilibrating condition.  This practice  may have the advantage of getting you to standard equilibration conditions faster and it will also clean your column.   A better alternative is to make the first run a blank run (or "preparation run") and then the next run can be your real analysis.  We prefer the second option because it should get you to the standard starting conditions more accurately.   However, often,  if we are in a hurry and the first option is quicker, well.....

After the Last Run of the Day
We store our columns in 50/50 methanol/water without any acid.  If you are using a salt, unlikely in LC/MS, wash your entire system, solvent bottles, HPLC, solvent lines, and column, into a non-salt containing solvent.   Salt may precipitate out and plug your HPLC or column or may cause corrosion.  Usually we flush with pure water first then leave the system in 50/50 methanol: water.  Some salts may precipitate out in high organic so an initial water wash is advised.  The 50/50 methanol:water solution helps to stop bacterial growth which can muck up your system.  Take care of your HPLC, it's the right thing to do!

   

HPLC: Tutorial

Introduction
The message of this tutorial is that reverse phase HPLC is simple.  Compounds stick to reverse phase HPLC columns in high aqueous mobile phase and are eluted from RP HPLC columns with  high organic mobile phase.  In RP HPLC compounds are separated based on their hydrophobic character.  Peptides can be separated by running a linear gradient of the organic solvent.  I often tell my fellow researchers to run the 60/60 gradient when chromatographing an unknown.  The 60/60 gradient means that the gradient starts at near 100% aqueous and ramps to 60% organic solvent in 60 minutes.  The majority of peptides (10 to 30 amino acid residues in length) will elute by the time the gradient reaches 30% organic.  To learn some of the simple principles of RP HPLC please read on. 
The HPLC

In most cases the HPLC you intend to use must be able to pump and mix two solvents.  This can be accomplished with one pump and a proportioning valve or by using two separate pumps.  Generally the pumping configuration is an aspect of the instrumentation that is transparent to the user.  Reverse phase chromatography can also be performed in a purely isocratic mode where the solvent conditions are held constant, this form of reverse phase chromatography can be carried out with a single pump.  Isocratic methods are used most often in a QC environment in which a single analyte has been extensively characterized and the compound is being run to confirm it's identity and to look for closely related degradation products.  If you do not own an HPLC here is a link to HPLC vendors and accessory suppliers.

HPLC Column Components and Specifications

a.      column dimension (size)

b.     particle size and pore size

c.      stationary phase

a.      Since columns are tubular, column dimensions usually take the following format, internal diameter X length (4.6mm X 250mm).  As a mass spectroscopist you will encounter columns ranging in internal diameter from 0.050 to 4.6 mm or even larger if you are performing large scale preparative chromatography.  For mass spectrometry a short reverse phase column will work nearly as well as a longer column and this is an important fact because shorter columns are generally cheaper and generate less back pressure.  Why is less back pressure important?  If a column runs at low pressure it allows the user more flexibility to adjust the flow rate.  Sometimes shorter columns are used to do fast chromatography at higher than normal flow rates.  In terms of length we routinely run 100 mm columns, however 50 mm or 30 mm columns may be adequate for many LC/MS separation needs.

b.     The most common columns are packed with silica particles.  The beads or particles are generally characterized by particle and pore size.  Particle sizes generally range between 3 and 50 microns, with 5 µm particles being the most popular for peptides.  Larger particles will generate less system pressure and smaller particles will generate more pressure. The smaller particles generally give higher separation efficiencies.  The particle pore size is measured in angstroms and generally range between 100-1000 angstroms.  300 angstroms is the most popular pore size for proteins and peptides and  100 angstroms is the most common for small molecules.  Silica is the most common particle material.  Since silica dissolves at high pH it is not recommended to use solvents that exceed pH 7.  However, recently some manufactures have introduced silica based technology that is more resistant to high pH, it is important to take note of the manufactures suggested use recommendations.   In addition the combination of high temperature and extremes of pH can be especially damaging to silica.

The stationary phase is generally made up of  hydrophobic alkyl chains ( -CH₂-CH₂-CH₂-CH₃ ) that interact with the analyte.  There are three common chain lengths, C4, C8, and C18.  C4 is generally used for proteins and C18 is generally used to capture peptides or small molecules.  The idea here is that the larger protein molecule will likely have more hydrophobic moieties to interact with the column and thus a shorter chain length is more appropriate.  Peptides are smaller and need the more hydrophobic longer chain lengths to be captured, so C8 and C18 are used for peptides or small molecules.  Here is an interesting note: Observations have been made that C8 columns are actually better for capturing smaller hydrophilic peptides, the theory here is that the longer C18 chains lay down during the early aqueous period of the gradient and the more hydrophilic peptides are not captured.  We use C8 routinely for all peptide work and this particular alkyl chain length works equally well if not better than C18 for all peptides.

Solvents

 

The reverse phase solvents are by convention installed on the HPLC channels A and B.  The A solvent by convention is the aqueous solvent (water) and the B solvent by convention is the organic solvent (acetonitrile, methanol, propanol).   It is important to follow this convention since the terms A and B are commonly used to refer to the aqueous and organic solvents respectively.  The A solvent is generally HPLC grade water with 0.1% acid.  The B solvent is generally an HPLC grade organic solvent such as acetonitrile or methanol with 0.1% acid.  The acid is used to the improve the chromatographic peak shape and to provide a source of protons in reverse phase LC/MS.  The acids most commonly used are formic acid, triflouroacetic acid, and acetic acid.  A 0.1% v/v solution is made by adding 1ml of acid per liter of solvent.  Triflouroacetic acid has been reported to suppress MS ionization and often mass spectroscopists  lower the percentage of TFA to 0.05 or even 0.02% without significant loss in chromatographic efficiency.  Some MS people add a small percentage of heptafluorobutyric acid (HFBA, pdf from Michrom) to acetic acid solvents or low TFA containing solvents to help improve peak shape.  Since modern mass spectrometers are very sensitive it is important not to use plastic pipette tips when adding acid to the mobile phase, always use glass.  In our work we use acetonitrile as our organic solvent.  We have heard that the best electrospray solvent is 30% methanol, 35 mM acetic acid.  We commonly use this solvent system for ESI MS infusion, but have found that acetic acid is an inferior acid for chromatographic peak shape.  Our preferred HPLC grade water, acetonitrile and methanol is purchased from Burdick and Jackson.  Our preferred TFA comes in 1 ml ampoules from from Pierce Chemical Company.
Our Preferred Solvent System for ESI LC/MS
A = HPLC grade Water, 0.1 % formic acid
B = HPLC grade Acetonitrile, 0.1% formic acid

Gradient

When chromatographing an unknown we normally use the following simple gradient to learn about the hydrophobic character of the unknown compound.  The % A in the gradient described below is implied.

We call this the 60/60 gradient, because we run from near 0% B to 60% B in 60 minutes.  Through experience we have noted that 90% of all peptides will elute from a C18 reverse phase column by 30% acetonitrile.  There may be a few really hydrophobic peptides that elute later that is why we take the gradient to 60% B.  You may even want to run this gradient to 80% at least once to see if you are getting everything off of the column.  You may ask why don't we start the gradient at 0% B?  As we talked about before, in 0% organic and in high aqueous, the very hydrophobic, long C18 alkyl chains in an effort to get away from the high aqueous environment mat down on the particle.  When these alkyl chains mat down they are inefficient at capturing the analyte so chromatographers in the know start the gradient with some small % of organic, 2-5%.

Flow Rate
It is important to use the correct flow rate for your HPLC column.  Below is a table with standard flow rates for easy reference.  If you are running a column with a different diameter than those shown in the table please review the maintaining linear velocity page to learn how to calculate the appropriate flow rate for your column.

Sample Preparation
The sample is normally reconstituted in the A solvent to maximize binding to the column.  The sample should not be dissolved in an organic solvent or it may not stick to the stationary phase.  The sample should not be dissolved in detergent containing solutions.  Some detergents may bind to reverse phase columns and modify them irreversibly.  In addition detergents preferentially ionize in electrospray mass spectrometry and can obscure the detection or suppress the ionization of the analyte.

Preformulation and Formulation Development

Preformulation and formulation development can deal with two challenges that might be considered as opposites:

·         Developing drugs with greater speed; and

·         Processes of drug development are growing more complex and time-consuming. From Lead to Clinical Trials

Let us imagine that, as a medicinal chemistry team, your lead has just been chosen. By outsourcing (drug discovery support) or in-house, you have got the materials to go through first toxicology and pharmacokinetic studies. Are you now ready to go to

clinical trials?

As a contract development organisation (CDO), Companies have seen several customers that have booked their clinical trials to a CRO (contract research organisation) and come to them to ask for some drug products to provide to the CRO. The step between the current Good Manufacturing Practices (cGMP) batch manufacturing of the active pharmaceutical ingredient (API) and the release for clinical trials, might be simple, but might be very complex also.

To address the question mark in Figure 1, consider preformulation and formulation development.

Preformulation

Preformulation is not only about stability and solubility data as shown in a lot of contract services supplier websites. Far from that, preformulation must be considered as an interface between the drug substance and the drug product.

According to the Product Quality Research Division of the US Food and Drug Administration (FDA), the goal of preformulation is to “investigate critical physicochemical factors which assure identity, purity of drug substances, formulatability, product performance and quality.”

Whatever the form may be, the end-use properties of the drug product are linked with:

·         Dose and release – what amount of the drug substance is needed in what time?;

·         Bioavailability and toxicity – drug performance level compared with side effects; and

·         Stability and shelf-life – to ensure quality and performance during storage.

Solubility determination, salt selection and pKa measurements are, of course, driven by dose. For instance, if the effective dose is 10mg and the drug solubility in water is 0.01mg/ml, it may be better to find a stable salt with a higher solubility than to inject one litre of solution.

Less known is the impact of crystal properties and polymorphism. If you have already experienced (or withstood) a polymorphic form appearing or disappearing, then you already know the major effect that crystal properties can have on solubilities, dissolution rate, toxicity, formulation process and so on. If not, what a lucky person you are! However, you must still be aware that the FDA recommends the characterisation of your drug substance by X- ray diffraction at the minimum, even in the pre-investigational new drug (IND) stage.

Particle size and surface area have an impact on dissolution rate. Usually, the higher the surface area, the higher the dissolution rate, but very fine particles can agglomerate, leading to caking and problems

during dispersion. By changing crystallization conditions on the drug substance, you may have a direct impact on solid handling during formulation step, solid handling needed both for oral forms (flowability) and parenteral (in the dosing hopper).  The effects of surface properties are less known, although they are of major importance for bioavailability. All interactions within the human body are driven by hydrophilicity and/or lipophilicity, depending on adsorption, cell absorption, membranes crossing, antibody interactions and so on. All the components of the ormulation are part of this game, but even in targeting delivery, the final step is the release of the active. The partition coefficient or surface tension for an oral form have to be taken into account.

A preformulation team needs experts in chemistry, analysis, chemical engineering and physical characterisations. A supplier in preformulation might be able to provide:

·         Physicochemical properties of the drug substance:

Ø      Solubility studies

Ø      Ssalt screening, pKa determination

Ø      Partition coefficient, hydrophilicity, lipophilicity

Ø      Crystallisation studies (impact on amorphous, particle shape, size and brittleness)

Ø      Polymorphism studies – identification, screening, relative stability (enantiotropy/ monotropy), process design and scale-up to ensure robustness of the obtained polymorphic form, dosage method of mixture;

·         Stability data:

Ø      Chemical stability, accelerated and stress studies

Ø      Thermal properties

Ø      Hygroscopicity (storage conditions)

Ø      Excipients and packaging compatibility studies; and

·         Early stage formulation:

Design composition and form according to specifications, dose and bioavailability. Depending on phase development, amount of drug available, the above preformulation studies will be adapted to obtain the right level of information according to the risk that the customer is ready to take.

In early stage, formulation might be the simplest option. However, even for the simplest  forms like solution and capsules, the right levels of stability data, analytical validation and standard operating procedure are needed for the release of the drug product for clinical trails. Fortunately, drugs must not be delivered to humans without some controls.

Let us give an example of a cytotoxic anticancer drug. For an oral form, capsules will be prepared by direct mixing, if possible, or granulation, if needed. During preformulation studies, excipient compatibility studies will have been carried out to choose rapidly the right ones to ensure stability. Bulk density of the API will have been checked, otherwise, depending on the API batch scale, bulk density might change even by a factor of 100%, leading to a powder that will no longer fill your capsules with the same amount of API.

At a minimum, polymorph identification on several batches will have been carried out also, although you will experience great differences in solubility if new polymorphic forms appear.

 For a parenteral formulation, if solubility and stability are correct, you will try a solution, otherwise you will have to go to lyophilisation. Cryomicroscopy and thermal analyses will be very useful to design the formulation and the lyophilisation cycle. Holding time of the solution before repartition, filtration conditions, reconstitution time on the lyophilised product, purity and stability will be checked to ensure a procedure that can be used for the manufacturing of the drug product for clinical trials.

It has to be noted that, with the new European Directive (May 2004) for Clinical Trials, batch manufacturing now has to be performed under cGMP rules and released by a qualified person.

For all forms, stability data is needed both on the API and finished product to give a shelf life for the drug at a minimum equivalent to the duration of the clinical trials. A supplier in formulation development might be able to provide:

·         Expertise and equipment for:

Ø      Oral forms – powders, granules, capsules, tablets, suspensions, emulsions, syrup

Ø      Parenteral forms – solutions, lyophilisates, nanoparticules, fine emulsions

Ø      Inhalation forms such as dry powder inhalation

Ø      Topical forms – semi-solids;

·         Innovation and scale-up knowledge in drug  delivery design – microencapsulation, spray drying, spray cooling, controlled release, phase inversion temperature emulsions;

·         Analytical method validation and stability according to ICH guidelines;

·         Regulatory (FDA, EMEA directives for clinical trials); and

·         Project management in order to handle all the time, cost and regulatory constraints for different internal and external suppliers – development team, manufacturing team, packaging, labeling and final release of the drug product.

Methodology

As a conclusion, methodology to balance speed, on one hand, and a complex and time-consuming process, on the other hand, will be:

1. awareness – be aware, or work with a supplier who is aware, of all the traps that might occur during development and scale-up, be aware of changing regulatory constraints and of risks;

2. expertise in development with people able to solve the problems that will occur and who have already experienced and overcome such trouble- shooting; and

3. communication and transparency between all the players so that the customer might be able to make the best choices and the best compromises