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Analytical methods employed on the pharmaceutical industry are mainly chromatographic procedures (e.g., HPLC and GC). With this in mind, the method development tips presented herein will focus on those chromatographic techniques.
HPLC
HPLC procedures fall into four basic categories—reverse phase, normal phase, ion exchange and gel permeation/gel filtration. Another common type of HPLC technique, ion-pairing, is a reverse phase procedure. Since about 80% of all HPLC work is done using reverse phase chromatography, the following discussion will be on it. In developing a reverse phase HPLC procedure, the following parameters should be considered:
• What analytes are involved? The goal of most analytical methods is to measure, quantitatively, the amount of Analyte(s) in a sample matrix. For raw materials such as active drug substances, the purity of the raw material is determined. For finished drug products or intermediates such as granulations, the content of active ingredient needs to be determined.
For raw material assays, sample preparation is generally fairly simple and straightforward, since the raw material itself usually represents the sample matrix in its entirety. The analysis of active ingredients in finished products, by contrast, requires a sample preparation that takes the matrix (inactive ingredients) into consideration. It must be determined, either by knowledge of the chemistry or laboratory studies, what interferences, if any, are due to sample matrix and how they can be overcome such that the active ingredient content can be accurately measured.
• What method of detection will be used? The method of detection should be chosen based upon the chemical structure of the target analyte(s). If the target analyte(s) are good UV absorbers, and there are no other UV-absorbing components in the matrix (placebo), then UV detection should be used. If the target Analyte(s) are UV active, and there are other UV active components in the matrix, then the chemist must be sure that the analyte or analytes of interest, are separated from placebo components, and from each other, if there are more than one active drug substances in the formulation being tested. If the target analytes are not UV active (carbohydrates for example), then other detection methods such as refractive index or electrochemical should be considered. It is recommended that UV detection be attempted first and that other detection methods be used only when absolutely necessary. Refractive index detectors, for example, require steady flow rates and constant temperature throughout the chromatographic system, thus increasing cost (column heater needed) and decreasing efficiency (long column equilibration periods). In addition, refractive index detectors can only be use with isocratic analyses. If an Analyte is a poor UV absorber such as a carboxylic acid, try using low wavelengths between 190 nm and 210 nm where almost everything has some UV activity. However, when using these low wavelengths, lack of matrix interference is extremely important due to potentially high background noise at low wavelengths. In addition, low UV wavelength chromatography limits the choice of mobile phase to solvents that have a low UV cutoff such as water and acetonitrile.
• Preliminary screening: An efficient way to speed up the methods development process is to perform a preliminary UV screening (by UV spectrophotometer) of all product components, both actives and inactives. Doing a UV scan from 360 nm to 190 nm on individual solutions of each products ingredient, allows the chemist to select the best wavelength for the analysis, one that gives the best sensitivity for target analytes (usually the UV maximum) and avoiding those that might be problematical in terms of interference. Additionally, if there is more than one active ingredient in the product to be tested, the separation of which is not possible or extremely difficult to achieve, and one active has a UV absorbtion maxima at a wavelength where the other active has no absorbtion, the analysis can be designed using a dual-channel detector, each channel set at the wavelength best suited for the respective analytes. This technique is known as wavelength masking.
• Selecting a column: Reverse phase columns come in wide variety of sizes and flavors—methyl, butyl, octyl, octadecylsilane, phenyl, amine, cyano, and others. Each has a functional alkyl silane bonded to silica, varying in their retentiveness and order of component elution, depending upon which functionality is involved. When selecting a column, start with an octadecylsilane (C18) column. If this column is too retentive (long retention times) but the desired separation is achieved, try a shorter chain silane such as an octyl (C8) column, or just use a C18 column that is shorter in length. If the desired separation can still not be achieved, try a different functionality such a phenyl, amino of cyano for example. Select a column length that results in the shortest run time without sacrificing resolution, peak shape quality and reproducibility. For basic compounds (many pharmaceuticals active ingredients fall into this category), try using base-deactivated versions of the above columns. This will improve peak shape and resolution.
• What mobile phase will be used? If at all possible, stick to simple mobile phases such a mixtures of water and methanol or water and acetonitrile.
Reverse phase HPLC mobile phases often consist of a mixture of water (water containing a buffer or other additive) and an organic modifier such as acetonitrile, methanol or tetrahydrofuran. If the target analytes are nonpolar (poor water solubility), separation can almost always be achieved with a mixture of water and methanol or a mixture of water and acetonitrile, or a mixture of all three. For more polar materials, the aqueous component of the mobile phase should be buffered at a well-controlled pH, based on the analyte’s pK value, so that the analyte is more organic and less ionic at the pH of the buffer. At a pH where the analyte if more ionic, it elutes rapidly because its solubility in the mobile phase is more dominant that its attraction to the column packing. Conversely, at a pH where the analyte is more organic and less ionic, its retention is enhanced due to its greater affinity to the column packing at that pH. A carefully selected mobile phase pH can be very effective in achieving separation of components with different pK values. The methods development chemist should choose mobile phase pH based on pK values of analytes coupled with actual laboratory experiments. Also, be sure to operate within the pH limits of the column.
Occasionally, it is not possible to use a pH where the Analyte becomes retentive. When this occurs, an ion-pairing reagent can be added to the mobile phase. These reagents are non-UV active, long-chain organic compounds containing acidic or basic functionalities, such as an aklysulfonic acid or an akly quaternary ammonium compound. The ion-pairing reagent forms an ion-pair with the analyte (kind of like a weak salt), thus slowing the migration of the analyte through the column, because the long alkyl chain of the ion-pair reagent is highly attracted to the column packing.
The last and final installment of Methods Development will describe how to design an HPLC separation and will address topics such as sample preparation, instrument parameters and GC methods development.
An analytical method is nothing more than a written list of instructions for performing a laboratory test procedure. A well-written analytical method can be performed by any chemist of average ability by simply reading the method without any further clarification. The method should list all the reagents and equipment needed and should provide a step-by-step procedure with a detailed explanation of calculations and results units. In addition, if spectra or chromatograms are generated, a sample spectrum or chromatogram should be included as part of the written method.
In addition to being well-written, first and foremost, an analytical method should work as intended and be practical in terms of functionality and efficiency. It should be robust enough to withstand slight variations in operating parameters, and rugged enough so that it can be used routinely and reliably by different analysts, on different days, in different labs and on different equipment.
The Method Development Process:
Developing an analytical method, whether simple or complex, is a process based on science but is often practiced as art. The novice chemist is stuck with science, because he or she does not have the experience to develop the science into an art. As chemists gain more and more experience and practice in the development of analytical methods, their intuitiveness will evolve to the point where the science becomes an art form. The normal evolution is as follows:
• Novice chemist—Pure science
• Intermediate chemist—Science plus some art
• Experienced chemist—Art with science intuitively embedded
In addition to science and art, a certain amount of luck is also involved.
The Billiards Approach:
A good pool player is always looking ahead to the next shot, scanning the table for position in order to maximize the number of consecutive balls that can be sunk before yielding to the other player. Analytical methods development, figuratively speaking, also requires looking ahead to the next shot.
The chemist who is developing a method must consider several factors before and during the process: the method’s intended use, who will be using the method, the analytical time cycle and cost.
The Method’s Intended Use:
Methods should be developed the “fit the bill” so to speak, without losing sight of the intended application. For example, a chemist whose expertise is in HPLC methods development might lean toward using HPLC when designing analytical test methods. However, is a simple titration or other basic procedure will achieve the same end, then the simpler procedure should be selected as the method of choice. Similarly, if a complex analytical procedure is required to perform a particular analysis, then it should be used, rather than simpler techniques that might not offer the needed specificity, accuracy or sensitivity.
Chemists involved in the methods development process can avoid overkill or under kill by asking, “What am I trying to accomplish?” For example, in a mixture of carboxylic acids (acetic, formic, malonic and succinic), if one needs to determine the total acidity for neutralization purposes, then the method of choice will be a simple acid-base titration. On the other hand, if the exact composition of the mixture must be known to compute mass balance or yield for example, then a more sophisticated method such as ion-exchange HPLC separation of the carboxylic acids must be selected.
The method development chemist must always stay focused on the intended application, as this will result in the development of analytical methods that are appropriate and sensible in terms of time and manpower allocation
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Who Will Be Using It?:
An elegant analytical method, one with many technique-intensive steps and novel twists, will be next to useless if its intended end user is untrained or minimally trained in analytical chemistry (i.e., a production operator or Q.C. inspector).
The methods development chemist must always keep in mind who will be the end user of the method. Suitability for its intended use is not the only criterion for an analytical method. It must also be designed around the skill level of the end user. A good example is water testing, specifically, the determination of chlorine content in water. An analytical procedure designed for an experienced chemist might involve an amperometric titration of chlorine with standard phenylarsine oxide solution, while a method designed for plant operator for instance, would probably be limited to using a swimming pool chlorine test kit.
Analytical Time Cycle:
An analytical method, in addition to its suitability and skill-level components, must have a time cycle that fits its intended application. A method that requires two (2) days to run because a long sample digestion or extraction procedure for example, is of little use to a Q.C. lab that needs results the same day. The methods development chemist must determine, up front, how fast a method needs to be prior to its design and development. The chemist can then design a procedure that can be executed within the required time frame.
Cost:
Another important consideration in designing and analytical method is its impact on laboratory costs. Part of the methods development process must include a consideration of cost in terms of the types of reagents selected, the quantities used, the required instrumentation, column selection (for chromatographic procedures), and glassware sizing. For example, when designing an HPLC method, methanol is much less expensive than acetonitrile; therefore, if either will work, then methanol should be the solvent of choice.
Similarly, when determining how to perform sample and standard preparations, use minimum volumes of solvents and the smallest possible glassware sizes without sacrificing accuracy. For example, if a method calls for a standard solution having a final concentration of 0.2 mg/mL in methanol, one could weigh 100 mg of standard and take it to 100-mL of methanol, resulting in a stock solution containing 1.0 mg/mL, which is then diluted 10.0-mL to 50.0-mL in methanol to give a final concentration of 0.2 mg/mL. The total volume of methanol used per preparation is 150-mL.
Alternatively, one could prepare a standard solution containing 0.2 mg/mL by weighing 50 mg of standard, taking it to 50.0-mL in methanol, and then dilute 5.0-mL to 25.0-mL. This procedure uses only 75-mL of methanol per standard preparation. If an analytical balance of sufficient accuracy is used, a 50 mg weighing is perfectly acceptable. If one were a big spender, the 0.2 mg/mL standard solution could be prepared by weighing 200 mg of standard and taking it to 1000-mL with methanol. The decision to do a single step preparation versus a weighing plus a dilution must be based upon both cost and accuracy.
In addition to cost considerations, the selection of reagents must also be driven by availability and safety. A chemical that is only available from one source, and/or not always available, should not be considered unless there is no alternative. Similarly, extremely hazardous materials such as carcinogens,, mutagens, and acutely or chronically toxic materials, particularly those whose safe handling is a burden to productivity, should be avoided whenever possible.
The methods development process should also consider the selection of instrumentation and, if the procedure is chromatographic, the selection of columns. If a method can be done by titration, why tie up an HPLC system unnecessarily? When selecting a chromatography column, select the cheapest one that works. This does not necessarily mean the cheapest column in price, although this is sometimes the case. It refers to the cheapest one in terms of overall time management. If a 150 cm C18 column works as well as a more expensive 250 cm C18 column for a particular separation, then buy the 150 cm column. On the other hand, if one has a choice between an $800.00 ion exclusion column that will perform a separation directly with minimal sample preparation, and a $250.00 C18 column that requires ion-pairing to effect the desired separation, the ion exclusion column might actually be cheaper in terms of overall sample throughput, labor and cost of reagents.
A detailed and technical treatment of method development in terms of chemical theory is beyond the scope of this article. Our goal is to provide guidance and suggestions for the practicing chemist, and to help jump-start the methods development process, particularly for chemists who are new to the world of methods development and analytical research and development in general.
Part II of this article will focus on the nuts and bolts of HPLC and GC methods development.
This installment will start the presentation of proper technique for executing a variety of general laboratory techniques and procedures. This article will focus on filtration, , and UV/Vis techniques, and centrifuging.
Filtration:
1. Gravity Filtration (Volumetric)
• Place a piece of folded filter paper (fluted filter paper) such as Whatman™ #2 filter paper into a suitable funnel.
• Wet the filter paper with the appropriate solvent. For example, if the sample is suspended in water, wet the filter paper with water.
• Place the funnel stem into the receiving vessel (flask, beaker, etc.) such that the bottom tip of the funnel’s stem touches the side of the receiving vessel. Tilt the receiving vessel if necessary.
• Pour the sample into the filter paper in portions, allowing each portion to filter before adding the next. Do not fill the filter paper more than about 90% of its capacity.
• After the last portion of sample has completely filtered though the paper, rinse the paper with several small portions of solvent, allowing each rinsing to drain completely before adding the next.
• Finally, remove the funnel from the receiving vessel, rinsing the outside of the stem with a small amount of solvent as the funnel is being withdrawn.
• If the filter paper tears or overflows during the filtering procedure, discard the sample and start over.
2. Vacuum Filtration
• Wet a piece of filter media (paper, Nylon®, Teflon®, etc.) with appropriate solvent. For example, if the sample to be filtered is aqueous, wet the filter media with water.
• Mount the filter media, such as a 0.45 micron filter disk, into a suitable vacuum filtration assembly.
• Place the filter assembly into the neck of a suitable vacuum filter flask and attach the sidearm of the flask to a source of vacuum (pump or aspirator), using heavy-wall vacuum tubing.
• Turn on vacuum and begin filtering the sample in portions, allowing about 90% of each portion to filter though before adding the next one.
• After the last portion of sample has been added to the filter assembly, and has filtered to dryness, rinse with filter assembly with several small portions of solvent, allowing each to filter completely before adding the next one.
• Break the vacuum by gently by slowing and gradually disconnecting the vacuum tubing from the sidearm of the vacuum filter flask.
• Remove and disassemble the filter funnel assembly, discard the media, and clean the apparatus for subsequent use.
• If the sample filters slowly, adding a filter aid to the sample, such as diatomaceous earth, may improve performance.
• As with gravity filtering, if the media breaks during filtration (this will usually only happen if it is installed improperly), start over.
3. Pressure Filtration
• Wet the filter media with solvent.
• Place the sample in the pressure assembly, close the assembly, and attach a source of pressure such as compressed air or nitrogen,
• Apply pressure until the sample has filtered completely.
• Disassemble the pressure unit, add rinse solvent, reconnect and filter the rinse. Repeat this procedure until desired rinsing is achieved. Three times is usually adequate.
• Disassemble the pressure filtration unit, either clean or discard the media, depending upon whether or not it is disposable, the clean the entire unit for subsequent use.
• NOTE: Pressure filtration is used when gravity of vacuum filtration is not feasible, such as with slurries containing carbon and with thick syrups.
UV/Vis Techniques
1. Turn on and setup the spectrophotometer as per the manufacturer’s instructions.
2. Prepare sample as per the analytical monograph for the samples at hand.
3. For visible range work, glass cuvettes are adequate.
4. For UV work, silica (quartz) cuvettes must be used.
5. Make sure that all cuvettes used for analytical work are matched pairs.
6. With single-beam instruments, zero with a cuvette containing the blank (solvent used for sample). Then measure each standard and sample, zeroing with blank after each measurement.
7. With double-beam instruments, place cuvettes containing blank is both the sample and reference beam, then zero the instrument.
8. Measure each standard and sample versus the blank by placing the cuvette containing standard or sample in the sample beam of the instrument.
9. Make sure that cuvettes are clean as any film or dirt can cause analytical error. Usually rinsing with solvent, followed by isopropyl alcohol and acetone will do the trick. Let the cuvettes air dry or suck dry with vacuum. Never use compressed air as it contains micronized oil particles that can contaminate the cuvettes. If all else fails, soak the cuvettes in hot concentrated nitric acid, followed by a thorough water and acetone rinse. NOTE: Never use chromic acid (CHROMERGE™), as it will dissolve the cement that holds the faces of the cuvette together.
10. The outside walls of the cuvette can be cleaned during use by exhaling lightly against the outside walls of the cuvette (same as fogging a window with one’s breath) and them rubbing the walls dry with a soft tissue or with lens paper.
11. Just prior to placing a cuvette into the instrument, hold it up to light and look though its optical path to make sure that everything looks clean and clear.
12. Do not exceed the range of the instrument. If a sample shows and absorbance that is over range, diluted the sample further, the rerun the analysis.
13. Do not overfill cuvettes. Filling them about three quarters full is adequate. As a general rule of thumb, if the liquid level in the cuvette covers the pathway in the instrument’s cuvette holder completely, there is enough sample volume for analysis.
14. Record all readings for standards and samples versus the blank.
15. Calculate results per the analytical monograph being used.
16. Clean and dry cuvettes thoroughly and put them away for subsequent use.
17. Shut down the instrument as per the manufacturer’s instructions.
Centrifuging
1. Examine the centrifuge to make sure that it is level and that the head (rotor) is securely mounted and tightened. Be sure to wear eye protection.
2. Place the centrifuge tubes or bottles containing the material to be centrifuged into the rotor’s tube or bottle holders. Always use an even number of tubes or bottles such that they are placed across from each other so that the weight of material in the centrifuge is evenly balanced. If only one sample is to be centrifuged, counterbalance it with a tube or bottle containing the same volume of plain solvent.
3. Make sure the centrifuge cover is closed and that any guards or locks are properly engaged.
4. Centrifuge at the specified speed and for the specified length of time.
5. If the centrifuge makes any unusual noises or banging sounds, turn it off immediately and correct the problem before continuing. Many centrifuges are equipped with a brake for emergency stops.
6. If the centrifuge “walks” while it is in operation, turn it off, rebalance the load and then resume operation.
7. After centrifuging for the specified time and speed, turn off the centrifuge and let it come to a full stop. Never remove sample from a centrifuge that has not come to a full stop. To do so may result in serious injury.
8. After the centrifuge has come to a full stop, remove samples.
The next article will present proper techniques for pH, conductivity and Karl Fisher titrations.
