The dissolution test has been under scrutiny in several areas: the quality-by-design initiative has called for the end to dissolution testing along with all end-product testing (1–3); there is a push for more clinically relevant specifications (4); the potential flaws in the hydrodynamic fluid flow patterns from the vessel and paddle interaction are being examined closely (5–8); and the use of the calibrator tablets has been questioned (9). 

The US Food and Drug Administration's quality-by-design and process analytical technology (PAT) initiatives urge manufacturers to know their drugs and drug products much more thoroughly than is the present practice. Nothing is more disheartening than a significant change in the dissolution results on stability of a Phase III product or on a release batch of a commercial product—especially when no assignable cause is forthcoming. The increased knowledge expected from PAT may prevent these "surprises," and that would be a welcome change. The dissolution test is sensitive to a nearly infinite number of parameters, from characterizations of the drug to formulation changes to, most importantly, manufacturing parameters. The dissolution test's ability to show changes in so many parameters is its power and its frustration. The power of the test outweighs the frustration for one simple reason: the dissolution test is the only test that has some degree of relevance to the drug's therapeutic effect 

Eliminating dissolution as an end-product test would be problematic from several angles. Can you be sure in-process testing has detected all of the many sources of potential change in the final product? How do you measure the stability of the finished product unless you test it at release and then over its shelf life? What is the value of eliminating a proven indicator of stability? 

The push for more clinically relevant dissolution specifications and methods is laudable, and in this effort, the method-development stage is particularly critical. Many a naïve manager has viewed the dissolution test as simple—until a problem occurs. Only then does the manager discover that the staff is too inexperienced to understand the test's nuances or sources of error (10). A separate dissolution-testing group is the optimal way to handle both dissolution-method development and routine testing. A group enables better training, accumulation of direct product experience, and useful collaboration. In addition, a separate laboratory devoted to dissolution testing will better avoid equipment problems stemming from vibration and other related issues. 

Finding the appropriate method and specifications, especially with the typical low drug solubility, takes time and resources. Cutting corners at this stage is very risky. The robustness and variability of the method should be examined thoroughly. Guidance on method development is available in the literature (11, 12), FDA guidances (13–15), a new proposed informational general chapter from the 

US Pharmacopeia: ‹1092› The Dissolution Procedure: Development and Validation

 (16), the American Association of Professional Scientists 

 Release and Dissolution Testing Focus Group, books (17, 18), and Web sites that offer chat rooms, bulletin boards, or interactive questions and answers (19–21).  

Variability should be examined early in method development. High variability is problematic, making trend analyses and F2 (similarity factor) calculations difficult. It is vital to isolate and understand the sources of variability. Observe the physical dissolution process for any anomalous stirring. The test should show gentle homogenous mixing. Note the hydrodynamic flow of the fluid and look for any coning (

 a concentrated gathering of excipients and drug under the paddle), tablet-sticking, air bubbles, or off-center placement of the dosage form, and examine the dissolution rate to see if  a correlation exists. If so, the method developers should make every effort to minimize this anomalous behavior. At the method-development stage, all aspects of the mechanical or physical dissolution test that can affect the results should be illuminated and minimized, so that if a dissolution test failure occurs later on, the failure can, with confidence, be attributed to some change in the dosage form.  

When the time comes to set specifications, the sponsor and FDA must collaborate to make the specifications appropriate. The specification must describe a very fine line, preventing bioinequivalent batches from passing, while not being so tight as to fail good (meaning fully effective 

) batches that may vary slightly. In some instances, a specification is borderline: over time, the product goes more and more to stage 2 retesting. Although batches may initially pass after retesting, this scenario may produce later failures and recalls. Again, special care should be taken to understand critical parameters and, in particular, the stability behavior of the product.  

In later phases of the product's life, method development and validation should include robustness of the method and examining the aspects of the test (as opposed to the product) that may influence the dissolution rate results. Typical parameters such as temperature changes, changes in media concentration, basket attachment type, paddle height, changes in media pH, and many other aspects should be altered within small tolerance ranges to see if the dissolution rate is sensitive to them. Other factors—such as the presence of air bubbles or dosage form position in the bottom of the vessel—should be examined. This process helps in understanding where the method is robust and where it may be overly sensitive. Developers can expand test-method instructions or modify the test itself. The importance of the method development and validation stage cannot be overemphasized. It assists in knowing and characterizing the product well and even predicting the 

 correlation is developed (22). Problems with variability, poor mixing, or fluid flow can usually be overcome with appropriate change in apparatus type, speed of rotation, sinkers, or even media choice.  

A discussion of the dissolution equipment is important: the dissolution rate is generated by the stirring mechanism interacting with the dosage form in the media. But always be aware that the dissolution equipment is a machine. The initial quality of the device and its subsequent care and maintenance will affect both operational reliability and product dissolution rate results. Almost any industrial process will produce a lemon occasionally, and any machine will wear out over time. The environment in which it operates will affect performance, and it must be running properly at all times. Current practice requires calibrator-tablet tests every six months to assess the performance of the dissolution equipment.  

Historically, calibrator tablets were developed because representatives from FDA, USP, and the Pharmaceutical Manufacturers of America (PhRMA) all agreed that vibration (internal and external) was affecting the dissolution results (23). In the late 1970s, the calibrator tablets were put in place and required in ‹711› 

Today, we still cannot assess vibrational effects except by calibrator tablet tests. A PhRMA study (24) assessing the value of the calibrator tablets, concluded that "some type of calibrator tablets should be maintained until enhanced mechanical calibration is further defined (

, establishing a definitive vibration tolerance)." Equipment manufacturers have diligently designed testers that have less and less internal vibration. Even well-designed equipment that is used for years for 1, 8, or even 24 hours per day will eventually show signs of wear, however. In addition, the external environment can subject the equipment to vibration from heavy foot traffic, nearby construction, or nearby equipment, to name just a few sources. We must also acknowledge that not all equipment on the global market is designed solidly. With no mechanical means to test vibration other than calibrator tablets, eliminating them from the equipment performance assessment raises great concern. It is well documented that vibration affects the dissolution results (25–29); in some cases, vibration biases the results high, giving a false passing result. The consequences of false passing results should be of great regulatory concern. 

Vessel asymmetry also is detectable by calibrator tablets. The glass dissolution vessel is not made from a mold but from a combination of individual hemispheres shaped from standard tubing (30). The irregularities in the vessel shape can cause a change in the fluid flow and change the dissolution results. FDA laboratory scientists pointed out this situation in a 1982 publication (31). Since then, other publications and practical laboratory experience have substantiated the finding (32). At this time, no available mechanical means of detecting flaws in the vessel design exists, although there may be some devices on the horizon. Until then, the calibrator tablets are the only appropriate tool for detecting this problem. 

Some recent articles suggest that new apparatus for dissolution testing may give less variability and more homogenous mixing, and might even produce better correlations with 

 performance of the product (33, 34). New technology has added to the utility of the dissolution test. Fiber optics have increased the automation of on-line testing. Premixed media also increase test efficiency. With novel dosage forms, the other official apparatus (3, 4, and 7) are becoming more suitable as are their modifications. Some performance tests for unique dosage forms may not use the official equipment at all; this is fitting and should not be resisted if the advantages are truly apparent. For immediate-release and extended-release dosage forms, however, Apparatus 1 and 2 can often provide appropriate methods with special care and study during the method-development stage. Approximately 700 compendial tests use the present apparatus for very large numbers of product brands. And, new products are constantly being approved with dissolution methods using Apparatus 1 or 2. The investment of resources and scientific data backing these apparatus is indisputable. Newly designed equipment must go through the same rigorous qualification and will be sensitive to the same parameters that affect the present equipment. Industry will resist a move away from the equipment it already owns. And the regulatory agencies have many times discouraged, from the podium, the proliferation of new equipment types.  

Though these arguments may appear to support the 

, this is not the case. A more thorough understanding of active pharmaceutical ingredients and finished drug products in the early development stages would undoubtedly benefit the industry. More careful training and analyst experience are of paramount importance to minimize sources of variability and maximize sensitivity to critical parameters during the method development stage. New equipment that significantly adds to generation of a proper 

 release test is a worthy endeavor. Until appropriate mechanical means to detect vibration and vessel asymmetry are created, calibrator tablets are our best tools, though a search for better ways to characterize the equipment should continue. 

The American Association of Pharmaceutical Scientists' 

 Release and Dissolution Testing Focus Group will sponsor a workshop May 1–3, 2006, in Arlington, Virginia. 

 is the president of V.A. Gray Consulting, Inc, Hockessin, DE.	

1. J. Woodcock, "The Concept of Pharmaceutical Quality," 

2. A.S. Hussain, "Quality by Design: Next Steps to Realize Opportunities," paper presented to the US Food and Drug Administration Advisory Committee for Pharmaceutical Science: Manufacturing Science Subcommittee, Sept. 17, 2003. 

3. A.S. Hussain, "Biopharmaceutics and Drug Product Quality: Performance Tests for Drug Products, a Look into the Future," paper presented at the USP Annual Scientific Meeting: The Science of Quality, Iselin, NJ, Sept. 26–30, 2004. 

4. H. Zhang and L. Xu, "Dissolution Testing for Solid Oral Drug Products: Theoretical Considerations," 

5. P.J. Missel, L.E. Stevens, and J.W. Mauger, "Reexamination of Convective Diffusion–Drug Dissolution in a Laminar Flow Channel: Accurate Prediction of Dissolution Rate," 

6. J. Kukura, J.L. Baxter, and F.J. Muzzio, "Shear Distribution and Variability in the USP Apparatus 2 Under Turbulent Conditions," 

 "Sensitivity of Dissolution Rate to Location in the Paddle Dissolution Apparatus," 

"Evaluation of Dissolution Hydrodynamics in the USP, Peak and Flat-Bottom Vessels using Different Solubility Drugs," 

9. L. Buhse, "Measuring and Managing Method Variability," paper presented to the FDA Advisory Committee for Pharmaceutical Science: Manufacturing Science Subcommittee, Oct. 25, 2005. 

10. V. Gray, "Identifying Sources of Error in Calibration and Sample Testing," 

11. B.R. Rohrs, "Dissolution Method Development for Poorly Soluble Compounds," 

"Dissolution Testing of Poorly Soluble Compounds," 

Dissolution Testing of Immediate Release Solid Oral Dosage Forms

(FDA, Center for Drug Evaluation and Research [CDER],Washington, DC, 1997). 

Extended Release Oral Dosage Forms: Development, Evaluation, and Application of In Vitro/In Vivo Correlations. Guidance for Industry

Waiver of In Vivo Bioavailability and Bioequivalence Studies for Immediate-Release Solid Oral Dosage Forms Based on Biopharmaceutics Classification System. Guidance for Industry

 Informational General Chapter ‹1092› "The Dissolution Procedure: Development and Validation," 

 (Dissolution Technologies, Hockessin, DE, 2004).  

 (Taylor and Francis, Boca Raton, FL, 2005).  

22. R.S. Uppoor, "Regulatory Considerations for 

23. A.C. Sarapu, A.R. Lewis, and M.F. Grostic, "Analysis of PMA Collaborative Studies of Dissolution Test Calibrators," 

24. PhRMA Dissolution Calibration Subcommittee. "Dissolution Calibration: Recommendations for Reduced Chemical Testing and Enhanced Mechanical Calibration," 

25. W. Beyer and D. Smith, "Unexpected Variable in the 

 Rotating Basket Dissolution Rate Test," 

26. W. Hanson, "Solving the Puzzle of Random Variables in Dissolution Testing," 

27. C.C. Collins, "Vibration: What Is it and How Might it Effect Dissolution Testing," 

 "Fine Tuning of the Dissolution Apparatus," 

29. B. Crist and D. Spisak, "Evaluation of Induced Variance of Physical Parameters on the Calibrated USP Dissolution Apparatus 1 and 2," 

30. P. Scott, "Geometric Irregularities Common to the Dissolution Vessel," 

 "Systematic Error Associated with Apparatus 2 of the USP Dissolution Test II: Effects of Deviations in Vessel Curvature From that of a Sphere," 

Articles

Though dissolution testing has been under scrutiny, it is still a powerful test method.

Challenges to the Dissolution Test Including Equipment Calibration

Scientists use several common techniques to analyze pharmaceutical solids. These methods, which include single-crystal X-ray diffraction, X-ray powder diffraction, differential scanning calorimetry, thermal gravimetric analysis, infrared, and Raman spectroscopy, have several advantages for the analysis of polymorphic drug forms and formulated products, but, in some cases, they can have some disadvantages (see sidebar, "Pros and cons of techniques for pharmaceutical solids analysis").

Solid-state nuclear magnetic resonance (SSNMR) spectroscopy has several advantages over these techniques. First, it is nondestructive and noninvasive. If little sample is available for analysis, scientists can acquire an SSNMR spectrum  and return the sample for any further testing. SSNMR spectroscopy can be used on bulk drugs and on all types of drug formulations (

 tablets, capsules, suspensions, creams, and transdermal formulations) with little to no sample preparation. SSNMR spectroscopy is uniquely suited for testing drug formulations because the NMR resonances for most pharmaceutical excipients occur in a narrow range of the NMR spectrum. Thus, it is easy to distinguish the excipient from the active pharmaceutical ingredient (API) NMR resonances. Spectral subtraction can even be used to eliminate excipient peaks from the spectrum.  

In addition, the technique can be used to study inclusion compounds such as those with cyclodextrins. Furthermore, the NMR spectrum can be used to examine host–guest interactions by monitoring changes in the NMR peak position and dynamics associated with a given nuclear site. 

Moreover, SSNMR spectroscopy is quantitative and selective. Because NMR spectroscopy is inherently a quantitative technique (

 signal intensity is relative to the number of nuclear sites at that specific resonance frequency), SSNMR spectroscopy also can provide quantitative information if done properly, as will be discussed later in this article. This technique can quantify mixtures of polymorphic crystalline forms, or of crystalline and amorphous materials. Analyses can all be performed without the need for pure forms or a standard curve. 

 Pros and cons of techniques for pharmaceutical solids analysis.			

The method also is selective; each NMR resonance seen in the NMR spectrum is caused by a specific nuclear site. Most often, 

C NMR spectroscopy is used, but many other nuclei (

H) can be used, depending on what will be studied. NMR spectroscopy is unique because it can monitor a specific nuclear site. For example, it can be used to look for changes in the local chemical environment that may be caused by drug–excipient interactions or changes during the formulation process, along with the formation of different polymorphic or amorphous forms. In addition to the technique's selectivity, isotopic labeling of low natural abundance nuclei such as 

O can be used to increase the sensitivity of a specific nuclear site. An example will be presented later in this article. 

SSNMR spectroscopy also can be used to examine the structure of the material and its associated dynamics. For crystalline materials, one can determine the number of crystallographically inequivalent sites in the unit cell. It also can detect conformational changes and hydrogen bonding to some extent. Other, more advanced, two-dimensional techniques such as rotational echo double resonance can be used to measure distances (1). For amorphous materials, SSNMR spectroscopy can be used to examine the degree of disorder because various processing techniques (

 lyophilization, spray drying, melt-quench, cryomilling) can vary the overall degree of disorder in the sample.  

Sometimes the system's dynamics also can be used to investigate other aspects of the drug being studied. For crystalline polymorphic forms, some forms may be less stable than other forms, which could be caused by the overall molecular mobility. Correlations between the overall mobility or stability of polymorphic forms can be made by measuring the spin-lattice relaxation times (T

). If a new form was discovered, the relaxation time could be measured to yield an early approximation of its overall stability. Sometimes, the same polymorphic form from different lots can exhibit different properties (

 stability, dissolution rate) which could be caused by the degree of crystallinity in the sample. The presence of defect sites or less-crystalline domains in the sample may not be observed in X-ray powder diffraction. This is not the case with SSNMR spectroscopy. These sites provide another avenue for the relaxation process of the spin states in the sample. Thus, a sample that has a lower degree of crystallinity would exhibit a faster spin-lattice relaxation time (T

), which may vary by less than a second to several seconds. This technique is not limited to analyzing only the bulk drug. It also can be applied to formulations. Changes in the overall dynamics of the drug during formulation could screen out potential drug formulations because of the increased mobility observed, possibly yielding a final candidate faster. 

Despite its advantages over other common techniques, SSNMR spectroscopy also suffers from some disadvantages. It often requires a lot of expertise in the technique to run it properly. And, the equipment can be expensive, though most narrow-bore solution NMR spectrometers can be adapted to perform SSNMR work. Another obstacle is that the experiments are not routine (

 the experimental parameters used must be determined for a specific compound before running the NMR experiment). Automation is difficult because the sample often fails to spin when using an automatic program and requires human intervention. Compared with the techniques listed in the sidebar, "Pros and cons of techniques for pharmaceutical solids analysis," SSNMR spectroscopy is the least sensitive and requires a large amount of sample to generate an adequate spectrum when studying low natural abundance nuclei such as 

Analysis times for SSNMR experiments also can be problematic because they can range from a couple of minutes to a few hours, to a couple of days or more, depending on the sample being analyzed and what type of NMR experiment is used. Peak assignment in the SSNMR spectrum can be challenging because multiple peaks could be present for a single nuclear site or they may be overlapping peaks. Another problem is that the chemical shifts in the solid state can often differ as much as 10 ppm from the solution NMR values. A current review article shows many applications of this technique to the analysis of pharmaceutical solids (2).  

Originally discovered in 1945, NMR involves transitions between nuclear spin states that become nondegenerate when placed in a magnetic field. The transitions resonate when an electromagnetic field at a specific frequency (

 based on the nucleus and magnetic field) is used.  

In general, nuclei have a spin quantum number (I) which may be an integer, half-integer, or zero (not NMR active). A nucleus with integral or half-integer spin has an associated magnetic moment. When placed in a magnetic field, a torque is produced  to align the magnetic moment with the magnetic field. Because this effect cannot occur, the torque produces a rotation about the axis of the magnetic field at a specific frequency of: 

in which γ is the electromagnetic ratio for the given nucleus and 

 is the magnetic field strength. If a second field, 

, that is static and small is applied to the system, the nuclei nutate or wobble slightly in their orbits. But if the 

 field oscillates at the same frequency as the nuclei, then the field will appear to be static to the nuclei and transitions between the spin states will occur. NMR spectroscopy is so powerful because each nucleus in the molecule is shielded by the surrounding electrons, so each nucleus sees an "effective field" that will change its resonance frequency. The frequency changes observed for the peaks in a molecule are reported in ppm to account for the differences in all of the magnetic fields that are used:  

(shift in hertz [Hz]) ÷ (spectrometer operating frequency [Hz]) × 10

Two problems for performing NMR spectroscopy in the solid state are dipolar coupling and chemical shift anisotropy. Dipolar coupling arises because of the sample's rigid nature. The equation for dipolar coupling is: 

for which γ is the magnetogyric ratio for the specific nucleus (A or X), 

 is the permeability constant, and θ is the angle between the static magnetic field and the spin pairs.  

in these equations. If a nucleus has both a high magnetogyric ratio and short internuclear distance, the dipolar coupling appears very large. For this reason, proton NMR spectroscopy is not performed in the solid state. For similar reasons, natural abundance also must be considered. With 

C at ~1% natural abundance, the probability of having two 

C nuclei next to each other in the molecule is very small, and thus only the heteronuclear dipolar coupling must be addressed. Normally, this dipolar coupling is ~50 kHz and is the strongest for CH and CH

; and much weaker for quaternary carbons. To solve this problem and reduce or eliminate the dipolar coupling, a decoupling pulse is used. The decoupling pulse in SSNMR spectroscopy is similar to that used in solution NMR spectroscopy, but as much higher power (

 typically a few hundreds of watts). During the acquisition of the free-induction decay (FID), the radiofrequency (RF) amplifier for the nucleus to be decoupled is turned on. This causes the spins to be flipped rapidly between spin states, thus stopping the dipolar-coupling spin-exchange process and thereby narrowing the resonances (see Figure 1). 

Chemical shift anisotropy (CSA) is another main problem. The NMR spectrum of a solid sample can be very broad because of the individual crystallites' multiple orientations in the magnetic field. The equation for chemical shift anisotropy is: 

in which σ is the observed chemical shift, σ

 is the anisotropic chemical shift, and θ is the angle between the shielding vectors and the magnetic field. To get high resolution NMR data in the solid state, only the isotropic chemical shift must be obtained. For this to occur, the anisotropic portion must be zero and can be accomplished by setting the angle to 54.7°. By rapidly spinning the sample about this axis (

 magic-angle spinning), the anisotropic portion cancels out thereby giving high-resolution spectra (see Figure 1). Spinning speeds can be approximately a few kHz to 35 kHz or more (

 180,000–2,100,000 RPM or more). The 54.7° angle is also the angle of the body diagonal of a cube. By spinning about this angle, each crystallite spends an equal amount of time in the 

 dimensions, thereby averaging out the crystallites' multiple orientations in the sample. If the spinning rate is not fast enough, artifacts, called s

, will appear at integral factors of the spinning speed (see Figure 1, only the peaks at ~17 and ~133 ppm are real). Based on the spinning rate compared with the width of the static NMR powder pattern, a certain percentage of the main peak intensity will be lost in the spinning sidebands. This point is important if quantitation is to be performed. 

Figure 1: Comparison of the 13C NMR spectra of hexamethyl-benzene acquired using several techniques. DD is dipolar decoupling, MAS is magic-angle spinning, and CP is cross polarization.			

The spinning sidebands can be eliminated in two ways. First, the sample can be spun faster than the width of the static powder pattern, which could be as high as 50 kHz or more. Second, a special pulse sequence can be used to cancel out the sidebands. More information about this process will be presented later in this article. 

C SSNMR spectroscopy, the recycle delay between transients can range from a few seconds to an hour or more. For this reason, cross polarization (CP) is often used.  

C CPMAS NMR spectroscopy, relaxation times are not dictated by the 

H relaxation, which is much shorter in most cases. A typical CP experiment consists of four main steps: generate the magnetization on the abundant nuclei (

H), transfer this magnetization to the dilute nuclei (

-axis by applying a π/2 or a 90° pulse. This procedure is followed by another pulse, which is sometimes termed the contact pulse, contact time, or the HartmanâHahn matching condition.  

The process of transferring the magnetization from the abundant to dilute spin is accomplished through the use of the heteronuclear dipolar interactions by a process of spin exchange. This will only work if the nuclei are at the same resonance frequency. Because the main magnetic field cannot be changed, the 

 field is applied at the resonance frequency of the specific nuclei, the nuclei will precess as if they were the only applied field. For 

, the HartmanâHahn condition is achieved.  

Often, the duration of the contact pulse must be optimized because two main factors affect the signal intensity: the cross polarization time constant (T

) and the proton spin-lattice relaxation in the rotating frame (T

). Initially, the magnetization builds up because of the T

 will cause the magnetization to decrease. Thus, a contact time period must be chosen that allows for the maximum magnetization transfer or maximum signal intensity. Not all samples will exhibit the same CP profile. Some samples may have a very fast T

 while others are slow. The same scenario applies to the T

. Thus, quantitation by CPMAS NMR spectroscopy must be performed very carefully. Another benefit of CP is that the sensitivity is increased by a factor based on the magnetogyric ratios of the two nuclei. The factor is 4 for 

A couple of advanced CP techniques such as variable amplitude cross polarization (VACP) and ramped CP also can be used. These techniques are needed because of the rapid spinning rates that are used in modern SSNMR systems. Because CP transfers magnetization through dipolar couplings, the dipolar couplings are partially averaged at fast spinning rates (

 the equation for dipolar coupling has the same angular dependence as the chemical shift anisotropy [3cos

  θ – 1]). This will lower the overall CP efficiency, hence yielding lower sensitivity. In both VACP and ramped CP, the intensity of the B

 field is varied, thereby transferring the magnetization that would have been lost in the spinning sidebands. Figure 2 offers a visual representation of the standard CP pulse sequence along with the VACP and ramped CP pulse sequences. 

Figure 2. Schematic representation of cross polarization (CP) pulse sequences. X) X channel (i.e., 13C, 15N), a) standard CP, b) ramped CP, c) variable amplitude CP. 1 is 1H 90Â° pulse, 2 is the contact pulse, 3 is the 1H decoupling pulse. 			

Another pulse sequence addition is that of total sideband suppression (TOSS). With complex NMR spectra, the presence of spinning sidebands can cause problems for determining which peaks are real and which are artifacts without conducting multiple spinning speed studies. The sequence consists of four 180° pulses in which the sideband phases are modulated. The times between the pulses—based on the rotor period (τ

)—are set so that the centerband is refocused and the sidebands cancel. A visual representation of the pulse sequence is shown in Figure 3 with an example spectrum. 			

This pulse sequence has a few disadvantages. The sideband intensity is not refocused into the centerband. If the spectrum was acquired at a low spinning rate, the centerband intensity can be greatly reduced. Another problem is that if the four 180° pulses are not perfect, then additional artifacts will be present in the  spectrum.

	Table I: 13C NMR chemical shifts and associated assignments.			

With SSNMR spectra, there are some general regions that can be used to aid in the assignment of a spectrum (see Table I). For the drug itself, the spectra can vary, but they mostly fall in the same regions because of the common functional groups. Most often, the API consists of aromatics, aliphatics, carboxylic acids, and heteroatoms. Excipients usually will not have any aromatics, aliphatics, or carboxylic acids, thus allowing for the easy identification of these elements in the NMR spectrum (see Figures 4 and 5). 

This article discusses the advantages and disadvantages of using solid-state NMR spectroscopy for the analysis of pharmaceutical solids.

Solid-State Nuclear Magnetic Resonance Spectroscopy for Analyzing Polymorphic Drug Forms and Formulations

Drug manufacturers invest a tremendous amount of time and money to identify, quantify, and minimize impurities related to their drug products so that the US Food and Drug Administration can make informed decisions regarding drug product purity and safety. An area of increasing concern and scrutiny for FDA's Center for Drug Evaluation and Research (CDER) is the potential adulteration of drug products by extractable and leachable compounds that enter a drug from a container, closure system, disposable, or device. 

				Drug product containers and closures shall not be reactive, additive, or absorptive so as to alter the safety, identity, strength, quality, or purity of the drug beyond the official or established requirements (1).			

				FDA provides guidance for protection against extractables and leachables in various documents (2, 3).			

Therefore, the qualification and quality control of all components coming into contact with the drug formulation is an integral part of any FDA application process. 

Extractables and leachables issues often are not addressed up front and ultimately can cause regulatory delays for the drug manufacturer. The development of unique packaging, novel formulations and delivery systems, and drug-coated medical devices has exacerbated this issue because of the increasing opportunities for foreign materials to come into contact with drug products. In addition, the increasing popularity of single-use disposables such as filters, tubing, and bags for biopharmaceuticals can introduce unwanted extractables into the final product. (For the rest of the article, the word 

 will refer to container closures, labels, drug delivery systems, packaging materials, devices, disposables, and so forth). 

The development, validation, and testing of these components must be carried out under International Conference on Harmonization and United States Pharmacopeia guidelines in a laboratory that complies with current good manufacturing practices. These activities may be time consuming and require expertise and a wide array of analytical techniques. Drug manufacturers may not have the resources available or may want to keep these resources focused on development of new products. Therefore, it is common for drug manufacturers to outsource these activities to contract laboratories. 			

 are compounds that can be extracted from a component under extreme conditions such as the presence of harsh solvents or elevated temperatures. These compounds can contaminate the drug product. 

 are compounds that leach into the drug product formulation from the component as a result of direct contact with the formulation under normal conditions. Leachables are typically a subset of extractables. Sources of these compounds include plastic components, elastomers, coatings, accelerants, antioxidants, inks, and vulcanizing agents. Phthalates are one specific example. These carcinogens are added to plastics to make them more flexible and can be found throughout the manufacturing process and in packaging materials. Other examples are nitrosamines and polynuclear aromatic hydrocarbons (PAHs), which are classes of carcinogenic compounds found in rubber. Many drug products are distributed or administered in packages made of plastic and rubber components, and, therefore, phthalates, PAHs, or nitrosamines could potentially come into contact with the drug product and be passed on to the patient. Metered-dose inhalers (MDIs), dry-powder inhalers (DPIs), and nebulizers can be complex because they may be constructed from a myriad of plastic, rubber, and stainless steel components. Nevertheless, these devices have many advantages (

rapid absorption and onset of activity and reduced dosing) for effective drug delivery. 			

In summary, extractables must be controlled to the extent that components used are appropriate. Leachables must be controlled so that the drug products are not adulterated. 

For an extractables and leachables evaluation to be meaningful and successful, it must be a collaborative approach on the part of the drug manufacturer, component vendor, analytical laboratory, toxicologist, and regulatory agency. Each party has critical input and is a significant contributor to the overall process (see Figure 1). The laboratory can only perform appropriate testing if all relevant information is provided. 

Figure 1. The collaborative approach to studying extractables andleachables.			

It is critical that vendors provide data on their potential extractables so that the manufacturer can choose the appropriate components early in the process. Vendors often do not make extraction data available to drug manufacturers, however. Ideally, after the extractables study, a toxicologist reviews the data to assess risk and set maximum levels based on total daily intake, as defined by the product dosing. This part of the process is typically beyond the scope of the analytical laboratory. Specific extractables are chosen to be monitored in the components and as leachables in the drug product. At this point, the laboratory can proceed to the other phases of the study. 			

Single-standard, regulatory-accepted procedures are not available. Each project is unique. Designing a study for a vial is much different than one for an MDI composed of many parts. In addition, the formulation must be considered. Injection products pose a greater safety risk than topical products. Studies are carried out in phases (see sidebar, "Various phases of study") that may be separated by long periods of time, during which analysis, data reduction, and risk assessment are performed. Each phase typically is described in a protocol. 

Extractable characterization is carried out first and is probably the most critical because all other decisions and testing are based upon it. Lancaster Laboratories (Lancaster, PA) has attempted to formalize a consistent approach, which is described in more detail later in this article. The process starts with gathering information and continues with the profiling and characterizing the extractables. In general, preparation requires both nonpolar and polar solvents. The instrumentation used includes high-performance liquid chromatography–photo-diode array detection–mass spectrometry (HPLC–PDA–MS) for organics analysis; gas chromatography–mass spectrometry (GC–MS) for organics analysis; inductively coupled plasma–optical emission spectroscopy or mass spectrometry (ICP–OES or ICP–MS) for metals analysis; and sometimes ion chromatography (IC) for inorganics and ion analysis. These techniques are complementary and provide a wealth of information needed to profile the extractables and leachables that may come from the component. Mass spectrometry is used because it is a powerful tool that elucidates structure. Sometimes more advanced MS detectors such as time-of-flight (TOF) are used to obtain accurate mass information. It should be noted, however, that unambiguous identification is not always possible. This issue will be discussed later in the article. Once the extraction profile is established, it is crucial that the toxicologists review the data, perform risk assessment, and propose maximum levels based on the total daily intake defined by the product dosing. 

After extractables have been characterized and qualified, the analytical methods are optimized and validated for the compounds of concern. Then, these methods are used for analysis of the components. Typically, several batches of components are tested, and suitable specifications are proposed for controlling consistency in the quality of these materials. 

Once appropriate components are chosen, analytical methods for leachables in the drug product are developed and validated. Samples of the drug products, which have been in contact with the components for an extended length of time, are tested. In case such samples are not available, the drug product and component are stressed under appropriate conditions to generate the leachables. Note that the leachables may be the same compounds as those identified during extractable studies or their chemical identity may be different from the extractables because of drug product interaction. All major compounds and target compounds that do not have origins in the drug substance or excipients are identified and quantified. These goals can be accomplished by making a formulation in an inert glass container to exclude the leachables originating from the components. 

The remainder of the article will discuss this critical first phase.  

Protocol for extractable characterization

To develop an accurate risk assessment of compounds that can be extracted or leached from a given component, the component must first be exposed to extreme solvents and conditions to generate every potential extractable. The goal is to quantify and qualify these compounds if it is analytically possible. Lancaster Laboratories has been following a consistent protocol when applicable. The general protocol is described below and is modified based on the component and drug product to be investigated. 

For organics analyses, each component is prepared so that the maximum exposure of the component to the extraction solvent can be attained. 

recommends that the total surface area (both sides combined) to volume of solvent ratio be 60 cm

 :20 mL (4). The components often are cut or crushed to maximize surface area. They undergo a variety of solvent extractions, typically employing either a Soxhlet extractor or sonication. For example, components may be exposed to hexane (

 aqueous) and heated for a specified amount of time. After extraction, internal standards are added to monitor system performance and to define lower detection limits for the analyses. 

For metals analysis, samples are prepared for ICP by placing a known amount of the component in a beaker and then adding deionized water and concentrated nitric acid. The sample is covered and heated at specified temperatures for a specified amount of time. Then, the samples are diluted into deionized water and analyzed by ICP–OES or ICP–MS. 

For each set of preparations, blank solutions containing the solvents without the component are prepared and analyzed in the same manner as the test samples. This process ensures that no contamination from the labware or reagents is present that could be interpreted as an extractable. Postextraction steps may include filtration, concentration, and exchange of solvent to improve detection. 

Hexane and alcohol extractions are analyzed by GC–MS. Blanks spiked with internal standard are first injected five times, and the percent relative standard deviation (% RSD) is determined to ensure the system is operating properly. In addition, the response is used to establish the detection limit. The mass spectra of detected extractable compounds are compared with the NIST98K database for identification. Although the database is used to assist in identifying the extractable components, the identification of each component cannot be guaranteed. All extractable peaks are quantitated on the basis of the average area response for the internal standard, assuming equivalent response factors. 

Water and alcohol extractions are analyzed by HPLC. The HPLC system is configured with PDA and MS detection and the mobile phase is water–acetonitrile or methanol. The PDA is used to screen the samples. Again, blanks spiked with internal standard are first injected five times and the % RSD is determined to ensure the system is operating properly. The response is used to establish the detection limit. The MS is set first in the electrospray positive mode, then the negative mode, and finally chemical ionization in the positive mode. 

The identification of the extractable compounds is attempted based on the molecular weight of the compound and compared to the molecular weight of known chemical compounds. The industry currently has no comprehensive databases for assistance in identifying liquid chromatography (LC)–MS-generated mass spectra. If a compound cannot be identified, an attempt is made to identify a class of compound for the extractable. Sometimes an accurate mass determination can be made using an LC–MS TOF. All extractable peaks are quantitated based on the average area response for the internal standard, assuming equivalent response factors. 

The acid-digested sample is analyzed by ICP–MS. Once digested, the sample can be analyzed for more than 25 elements simultaneously with detection limits in the part-per-trillion to part-per-billion range. 

As mentioned previously, mass spectrometry is a powerful tool for structural elucidation. Still, it is not always possible to make an indisputable identification of every peak (or compound) detected. Each of the different mass spectrometry techniques has its advantages and disadvantages. GC–MS data consist of fingerprint patterns that can be compared with large databases of organic compounds. Identification often can be performed with a high degree of confidence, and certified standards can be purchased for confirmation. The compounds amenable to GC, however, only make up a minority of the possible organic compounds that may be present in a component. Furthermore, some classes of compounds such as alkanes yield very similar fingerprint patterns or fragments, and thus a specific molecular entity cannot be identified. 

LC–MS data typically only provide a few ions, not a fingerprint pattern as those observed with GC–MS. Other advanced techniques such as tandem mass spectrometry (MS–MS) and TOF can provide more information such as molecular fragments and accurate mass. Database matching, however, is not achievable as it is with GC–MS. Orthogonal approaches such as nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) spectroscopy also can be used to try to solve this sometimes complex puzzle. 

At some point, the toxicologist should perform a risk assessment. These other analytical techniques can be time-consuming and expensive and do not always yield answers. Sometimes the data must be looked at from a different perspective. For example, an extractable is detected at 50 ppb from a component sample exposed to a strong solvent at high temperature for an extended period of time. The drug product is formulated in an aqueous buffer, exposed to this component for a relatively short amount of time, and must be kept refrigerated. Therefore, the extractable would probably not leach into the final product. The effort put into method development, analytical testing and identification must be weighed against the risk. 

Qualification and quality control of components that come into contact with the drug formulation is an integral part of any US Food and Drug Administration application. Therefore, extractable and leachable issues should be investigated and resolved early in the process. For a successful study, it is imperative that the component vendors, laboratories, toxicologists, and the regulatory agency have open, effective, and timely communication. As new delivery devices, disposables, and medical devices are developed, it is certain that FDA will continue to demand information about these components so that the agency can make informed decisions on risk and safety. 

, is the director of method development and validation and biopharmaceutical services for Lancaster Laboratories, 2425 New Holland Pike, PO Box 12425, Lancaster, PA 17605-2425, tel. 717.656.2300, fax 717.656.2681, 

(Office of Federal Register, Washington, DC, April 2005), Part 211.94, p. 128. 

Guidance for Industry: Container Closures Systems for Packaging Human Drugs and Biologics 

(FDA, Rockville, MD, May 1999), pp. 1–56. 

Guidance for Industry: Metered Dose Inhaler (MDI) and Dry Powder (DPI) Drug Products

 (draft, FDA, Rockville, MD, October 1998), pp. 1–65. 

United States Pharmacopeia 28—National Formulary 23

, (US Pharmacopeial Convention, Rockville, MD, 2005) p. 2269.  

Extractable and leachable issues should be resolved early in the drug manufacturing process. Open communication is imperative for a successful study.

Pharmaceutical Technology-02-01-2006

Challenges to the Dissolution Test Including Equipment Calibration

Solid-State Nuclear Magnetic Resonance Spectroscopy for Analyzing Polymorphic Drug Forms and Formulations

Identification and Risk-Assessment of Extractables and Leachables