Allen C. Templeton, PhD, is a research fellow at the pharmaceutical research and development unit at Merck Research Laboratories, West Point, PA 19486, tel. 215.652.9204, fax 215.652.2835.

Allen C. Templeton, PhD

Formulations for preclinical safety assessment studies are often prescreened by formulation scientists 

prior to use in animal models. By simulating the precipitation characteristics of a formulation in biorelevant media, the performance of a formulation can be predicted. The predictability depends on the sensitivity of detection and simulation of the mixing of the vehicle and biorelevant media. The authors describe the miniaturization of these types of experiments using a microfluidic chip system, combined with detection by optical microscopy. The results show the potential of microfluidics as a tool to help reduce compound requirements, time, and costs in formulation development. 

Dispersion experiments are heavily used in the pharmaceutical industry to screen potential oral formulations 

. The studies are run to rank how several formulations will perform prior to dosing new chemical entities in vivo. The experiment intends to mimic what occurs as the formulations are dosed orally and traverse through the gastrointestinal tract (1, 2). This experiment also is used to understand the mechanism of variability upon oral dosing. As a result, compound and animal resources can be spared when failed formulations are not pursued in actual animal studies. This experiment is particularly useful in drug discovery and development by saving time and resources while still advancing drug candidates. 

Microfluidic systems are evolving as useful tools for miniaturizing a variety of standard laboratory experiments (3). Many industries are using such systems, and more recently, the pharmaceutical industry is taking note (4). There are applications for microfluidics across the pharmaceutical industry ranging from scaling down the synthesis of challenging molecules to evaluating the effects of new chemical entities on biological systems (5).  

Microfluidic systems are of particular interest to the pharmaceutical sciences as tools for formulation development. Emulsion-based formulations are highly prevalent in the industry. Okushima et al. demonstrated that monodispersed double emulsions could successfully be generated with tunable control over the size and number of internal droplets produced (6). In this example, a specialized chip with hydrophobic and hydrophillic junctions was used to generate the various double emulsions. 

Liposomes also have been of interest to formulation scientists for several decades (7). Jahn et al. demonstrated the successful generation of liposomes on a microfluidics device (8). The liposomes generated were monodispersed, and the size of the particles was controlled by varying the flow rate. The mixing capabilities of the chip allowed liposomes to self-assemble in the channel, which would also allow for drug encapsulation and ultimately liposomal formulation preparation. 

Both of these studies used droplet-based microfluidics. The liposome-generation example used the microfluidic technique of hydrodynamic focusing, and the double-emulsion example used T-junction droplet generation (9). With respect to droplet microfluidics, the methods are complementary. In general, droplet-based microfluidic systems have the advantage of creating separated droplets that distinguish small amounts of reagents (10). T-junction droplet-generation systems are particularly flexible as several T-junctions can be placed in tandem, thereby allowing sequential reactions or mixing opportunities to occur. 

This article describes the use of a T-junction droplet microfluidics system to evaluate oral formulations of select model compounds by executing a simulated gastric fluid (SGF) redispersibility experiment directly on a microfluidics chip. This article describes preliminary proof-of-concept experiments. Morever, the results show the potential to save significant amounts of compound, animal resources, time, and cost in developing formulations for new chemical entities. 

The following materials were used in the study: naproxen (Chemical Abstracts Service [CAS] Number 22204-53-1, Sigma-Aldrich); naphthol (CAS Number 90-15-3, Sigma-Aldrich); perfluorodecalin (PFD) (CAS Number 306-94-5, Acros Organics), polyethylene glycol 400 (PEG 400, CAS Number 25322-68-3, Sigma Aldrich; Imwitor 742 (Sasol), and polysorbate 80 (CAS Number 9005-65-6, Tween 80, Fisher Scientific. 

Formulations were prepared at 50 mg/g concentration representing a 100 mg/kg dose in vivo when PEG 400 is dosed at 2 mL/kg. Model compounds naproxen and naphthol were fully solubilized at this concentration. In addition, naphthol was prepared at 100 mg/g in 1:1 Imwitor 742:Tween 80. The compound also was fully solubilized. SGF was prepared by dissolving 2.0 g of sodium chloride (NaCl) in 1 L of deionized water and adjusting the pH to 1.2 with 1 N HCl (hydrochloride acid).  

The microfluidics system was from Dolomite Microfluidics, and the experiments were run on a standard quartz T-junction chip with a channel size of 190 µm. The reagents were fed into the chip using three Dolomite Mitos Pressure Pumps (P-Pumps) with the pressure attenuated based on the viscosity of the liquids. The experiments were run at room temperature. After the experiments were completed, the chips were disassembled and transferred to either a Zeiss Axiovert 200M inverted microscope or a Nikon Eclipse E800 microscope, both equipped with a polarizing light filter to assess crystallinity. 

 Figure 1: Quartz microfluidic channel chip used in experiments as compared to the size relative to a penny and the schematic representation of the chip (top) and the naproxen formulation in polyethylene glycol 400 (PEG 400) dispersed as a plug in the carrier fluid perfluorodecalin (PFD) (bottom). The plug is optically distinguishable from the carrier fluid and also traveled freely through the channel.The microfluidic system used for this study was the Dolomite pressure-based droplet microfluidics system. The experiment required three Mitos-P pumps with one equipped with a three-way outlet head. A standard quartz channel chip was used, which was optically transparent and stable to organic reagents. 

 shows an image of the chip relative in size to a penny and also the schematic representation that is used throughout this article. In the T-junction schematic, the phase coming through Channel B would be considered the “dispersed” phase that would be dispersed within the “continuous” phase flowing though Channels A and C. 

The lines are supplied by the Dolomite P pumps, with the pressures adapted to the individual viscosity of each liquid and until suitable droplet size is achieved (droplets of B within the carrier fluid channeled through Channels A and C).  

The goal of these experiments was to see whether a typical redispersibility experiment could be performed on a droplet-generating microfluidics system. The redispersibility experiment involves preparing a formulation, either solution- or suspension-based, of a new chemical entity in a dosing vehicle and then dispersing the formulation into a biorelevant medium. Potential outcomes include the compound crashing out as either crystalline or amorphous physical phase or the compound remaining completely solubilized. If the starting formulation is a solution, when the compound of interest precipitates from the formulation vehicle, it is considered a less favorable formulation as this will render the active compound less absorbable in the intestinal barrier. 

The initial experiment performed was to generate a plug of a solution formulation of naproxen dissolved in PEG 400 within the carrier fluid, PFD, to verify that the viscous PEG 400 would travel through the channel of the chip, which proved successful (see 

). PFD was chosen because as a fluorocarbon, it is immiscible with PEG 400 and the aqueous-based SGF. In this experiment, the carrier fluid was plumbed through Channels A and C as depicted in 

, and the naproxen/PEG 400 formulation was brought in through Channel B. The naproxen/PEG 400 formulation was successfully “plugged” in the PFD carrier fluid and was visually distinct and traveled easily through the microfluidic channel. 

Figure 2: Benchtop-dispersed droplets of a naproxen/ polyethylene glycol 400 (PEG 400) formulation in simulated gastric fluid within the microfluidics chip channel separated with the perfluorodecalin carrier fluid. The image was taken under polarized light.Next, the same naproxen in the PEG 400 formulation was dispersed into SGF on the benchtop, and the resulting suspension was plumbed through the chip. The experimental design was the same in that Channels A and C were the PFD carrier fluid and Channel B was the suspension of the naproxen formulation dispersed (on the benchtop) in SGF. This experiment verified that plugs of a formulation suspension could travel through the channel of the chip. For visualization, the chip was removed from the microfluidics chip-holder system and examined with polarized light microscopy. The characteristic birefringence was observed through the quartz chip, suggesting that the naproxen precipitates from the PEG 400 as crystalline material (see 

 Figure 3 (a): Schematic representation of the dispersion experiment plan. The yellow channel represents the simulated gastric fluid (SGF); the pink channel the naproxen dissolved in polyethylene glycol 400 (PEG 400); and the grey channel the perfluorodecalin (PFD) carrier fluid. Figure 3(b) is the schematic representation of the second dispersion experiment plan. The SGF droplets were created first and subsequently mixed with the formulation. The yellow channel represents the SGF; the pink channel the naproxen dissolved in PEG 400; and the grey channel the PFD carrier fluid.Dispersing the naproxen in PEG 400 formulation into SGF directly on the microfluidic chip was attempted next. The design of the experiment is outlined in 

. The naproxen in PEG 400 formulation (see pink channel, Figure 3 [a]), SGF (see 

) were plumbed into three separate lines with the intention of generating droplets of dispersed formulation in SGF separated by the carrier fluid at the junction of the chip. This experiment was somewhat unsuccessful as the high viscosity of the PEG 400 formulation line caused significant backflow issues. The formulation precipitated; however, there was no controlled droplet generation. Looking at the chip under polarized light indicates the naproxen/PEG 400 formulation crashed out of solution generating a crystalline precipitate, which was a promising result; however, more control was desired. 

Figure 4. Droplets of the naproxen/polyethylene glycol 400 (PEG 400) formulation dispersed in simulated gastric fluid directly within the microfluidics chip channel, separated with the perfluorodecalin carrier fluid. The image was taken under polarized light.To achieve more control over the redispersibility experiment, the experimental design was changed. In the new experiment, SGF droplets (see 

) were first generated in the carrier fluid [see 

) and as they exited the chip, the exit line of tubing was replumbed into one of the bottom channels of the microfluidics chip. The second of the lower channels of the chip was plumbed with the naproxen in the PEG 400 formulation (see 

). This experiment proved successful as once the formulation was dispersed into the SGF droplets, the new combined droplets continued to travel through the channel. In addition, the timescale was appropriate to observe the precipitation of the naproxen into crystalline material before the newly formed droplets exited the chip (see 

Figure 5: Dispersion of the naphthol/Imwitor 742:Tween 80 formulation dispersed in simulated gastric fluid directly within the microfluidic chip channel. The image was taken under polarized light. Inset: nonpolarized light image.Having demonstrated that it is possible to perform a redispersibility experiment within a microfluidic chip, the final experiment aimed to differentiate between different formulations of a model compound. In this case, formulations of naphthol were prepared at 50 mg/g in PEG 400 and 100 mg/g in Imwitor 742:Tween 80. The difference in concentrations of these two formulations represents the difference in dosing volumes for the vehicles when administered at the same dose. The design of these experiments follows the schematic detailed in 

. The naphthol formulation in PEG 400 was dispersed first. As the formulation mixed with the SGF droplets and traveled through the channel, there was no precipitation event (data not shown). 

Imwitor 742:Tween 80 is a known self-emulsifying drug-delivery system (SEDDS) (11). SEDDS vehicles are typically comprised of oils and surfactants and solubilize highly lipophilic compounds. Upon reaching the aqueous environment of the stomach, the vehicle creates emulsions, which often boost in vivo exposure. When this formulation of naphthol was mixed with SGF droplets at the T-junction, the emulsifying nature of the vehicle was observed. This emulsion property is particularly evident when viewed under nonpolarized light (see 

 inset). When viewed with the polarizing filter on the microscope, it is clear that the naphthol compound was precipitating out as evidenced by the birefringence observed at the interface of the two solutions. The precipitation, however, was somewhat impeded by the emulsification of the Imwitor 742:Tween 80 vehicle, and no distinct crystal habit was formed (see Figure 5). 

Figure 6: Naphthol/polyethylene glycol 400 (PEG 400) formulation before and after dispersion into simulated gastric fluid (SGF) and naphthol/Imwitor 742:Tween 80 [1:1] formulation before and after dispersion into SGF.Having shown it was possible to disperse a formulation in SGF on a chip, a comparison experiment was conducted examining how the same formulations behave when dispersed in SGF on the benchtop. Standard SGF dispersion experiments were set up where the solution formulations of naphthol in PEG 400 and naphthol in Imwitor 742:Tween 80 [1:1] were dispersed into SGF at a ratio of 1:1. These experiments were run in small scintillation vials equipped with stirbars. 

 shows the formulation vials prior to and after SGF dispersion. The results of the traditional benchtop SGF dispersion experiment parallel what was observed in the microfluidic dispersion experiments. Upon dispersion into SGF, the naphthol in PEG 400 formulation remained a solution, and the naphthol in Imwitor 742:Tween 80 [1:1] formulation became cloudy. 

These experiments demonstrate the proof of concept that a dispersion experiment can be performed within a T-junction microfluidics chip system. When individual droplets of SGF were mixed with formulations at the T-junction, the model compounds either stayed in solution or precipitated out. When the compounds did precipitate out, information about the physical form of the precipitated chemical entity was obtained as the microfluidic chip is visualized under polarized light microscopy. 

As there are significant benefits, scaling down redispersibility experiments also was explored by other groups. Dai and Mansky (12, 13) describe a method using a 96-well plate that uses a small amount of compound to screen the solubilizing effect of several excipients once dispersed into simulated intestinal fluid [SIF]. The method is further validated by comparing the results of the miniaturized experiments to traditional formulation dissolution testing and actual in vivo studies (14). In addition, Gopinathan et al. (15) developed a 96-well plate based high-throughput formulation screening strategy. The company TransForm (16, 17) has developed similar high-throughput formulations screens, even extending to a 384-well plate. For these examples, the reagents are mixed by vortexing or sonicating the plate, or in some cases, heating to solubilize the initial compound. These examples illustrate the direction the pharmaceutical industry is headed with respect to formulation screening in the discovery space: using small amounts of compound to evaluate many different formulations. The experiments detailed in this article are complementary as they use microfluidic technology, specifically T-junction droplet generation. Although not as developed as the aforementioned examples, this method offers the potential benefit of in situ mixing that droplet microfluidics offers (18, 19). This feature would be beneficial when highly viscous formulations are evaluated and may be a better representation of how formulations are dynamically mixed in the gastrointestinal tract. Further, microfluidic technology has the potential to consume even less compound than what would be used in a 384-well plate experiment. 

Future experiments under consideration by the authors include a two-step SGF and fasted state SIF redispersibility study that would more closely mimic the transport of a compound from the acidic environment of the stomach to the more neutral pH (i.e., pH 6.5) of the small intestine. In addition, the system can be temperature-controlled, and the effect of temperature on these types of experiments may be explored. 

In summary, scaling down redispersibility experiments to the microfluidic scale has the potential to have significant impact on how formulations are screened in the pharmacuetical industry. Performing redispersibility experiments on a much smaller scale allows more formulations to be screened 

 using less material. When combined with more powerful detection techniques, the experiment also can provide more understanding of the behavior of the physical phase in the gastrointestinal environment. This information can help formulation development scientists identify and understand the most effective formulations for future in vivo experiments more efficiently. 

The authors gratefully acknowledge the New Technology Research License Committee of Merck & Co. for funding these experiments as well as Richard Gray, head of sales at Dolomite for helpful discussions. 

3. X. Casadevall i Solvas and A. deMello, 

7. V. Ravichandiran, K. Masilamani, and B. Senthilnathan, 

15. S. Gopinathan, A. Nouraldeen, and A.G.E. Wilson, 

16. C.R. Gardner, C.T. Walsh, and O. Almarsson, 

Nicole L. Hill*, PhD, is a principal scientist in the Basic Pharmaceutical Sciences Group at Merck & Co., 2015 Galloping Hill Road, Kenilworth, NJ 07033, tel: 732.594.2009, nicole_hill@merck.com. Todd Nelson, PhD, is an executive director in the Process Chemistry Group, Tim Rhodes, PhD, is a senior principal scientist in the Analytical Sciences Group, and Allen Templeton, PhD, is an executive director in the Basic Pharmaceutical Science Group, all at Merck & Co. Elizabeth Kwong, PhD, is a consultant with Kwong Eureka Solutions, and formerly a senior scientific director with the Basic Pharmaceutical Sciences Group at Merck & Co. Brian Farrer, PhD, is a principal scientist at Liquidia Technologies, and formerly a principal scientist with the Basic Pharmaceutical Sciences Group at Merck & Co. 

*To whom correspondence should be addressed. 

Submitted: Oct. 2, 2012; Accepted Nov. 12, 2012. 

Articles

The miniaturization of preclinical safety assessment studies using a microfluidic chip system and optical microscopy can help reduce compound requirements, time, and costs in formulation development.

Miniaturization of a Simulated Gastric Fluid Dispersion Experiment On a Microfluidics System

The control of moisture in pharmaceutical products is often necessary to prevent deleterious chemical and physical changes to a product. Moisture-driven changes in solid oral dosage forms can have severe adverse effects on pharmaceutical quality. Hence, several measurement approaches for product-moisture content currently are in routine use in the industry. Among the most common means to ascertain moisture levels in solid oral dosage forms are destructive loss-on-drying (LOD) or Karl Fischer (KF) titration techniques. An attractive augmentative approach for examining product moisture would be to directly determine the chemical potential of water in the sample by means of water activity (

), which is an arguably more fundamental indicator of the available water for the promotion of chemical, physical, or biochemical change (1–4). The chemical potential of a component in a mixture—such as water—is a fundamental measure of the "escaping tendency" or the availability of the component in the mixture, rather than the total moisture (which may include water that is bound or otherwise unavailable). 

				At typical storage temperatures and humidities for pharmaceutical solids (5–40 °C and <85% relative humidity), water behaves like an ideal gas, and the activity of water in a solid can be related to the headspace relative humidity by the following (5, 6):			

  is the partial pressure of water in the headspace, and P

  is the saturated vapor pressure of water at the temperature of interest. 

Thus, the relative humidity in the package headspace over a pharmaceutical solid at equilibrium can be directly related to the water activity and is an indication of the amount of water present with potential to cause undesirable changes in the pharmaceutical product.  

Currently, two different types of water activity instruments are commercially available: one based on a chilled-mirror dew-point technology to measure humidity and the other based on a polymeric thin-film capacitance sensor that is responsive to headspace moisture. In both of these techniques, the sample of interest is equilibrated in a sealed chamber until equilibrium between the solid and headspace moisture is reached. The chilled-mirror instrument consists of an optical reflectance sensor that detects the exact point at which condensation first appears at the mirror–headspace gas interface. This point is determined by directing a beam of infrared light onto the mirror surface and reflecting the beam of light to a photodetector, which detects reflectance changes upon initiation of condensation. A thermoelectric (Peltier) cooler helps to control the mirror temperature precisely, and a thermocouple attached to the mirror accurately measures the dew-point temperature. Both the dew-point and measured sample-surface temperatures are then used to determine the water activity.  

A less common approach involves capacitance measurements to obtain water activity results. A sensor made from a thin-film hygroscopic polymer relates the film capacitance to the headspace moisture content. Because this instrument relates an electrical signal to relative humidity, the sensor must be calibrated with known salt-solution relative humidity standards. In addition, for successful measurement, the sample and sensor must be at the same temperature. Although both of these instruments can be effectively applied to headspace moisture measurements, the nondestructive nature of frequency modulation spectroscopy (FMS) seemed to make the instrument ideal for exploration for examining the headspace moisture over pharmaceutical solids. The major shortcomings of the present techniques relate to the introduction of bias to the measurement and the inability to control measurement temperature over a wide range. Adding another sophisticated tool to the available arsenal of techniques to conduct critically important water activity measurements seemed to be worthwhile from the perspective of further refining the interface between measurement science and packaging research and development (7–9). 

 Figure 1: Principle and schematic of frequency modulation spectroscopy. Top figure (a) shows the frequency and intensity profile of a diode laser beam. Diagrams from left to right show an unmodulated, modulated with no absorption, and modulated with absorption by the upper sideband. Bottom figure (b) is a schematic diagram of an instrument for frequency modulation spectroscopy.			

Fundamentals of frequency modulation spectroscopy 

Water molecules in the gas phase absorb near infrared (NIR) light in a band of rotational transitions centered at 1400 nm. Absorbance measurements at this wavelength on typical NIR spectrometers using incoherent white-light sources lack the sensitivity to provide a useful measure of headspace moisture levels because of low-frequency lamp-intensity fluctuations. The measurement signal-to-noise (S/N) ratio can be vastly improved (100–10,000X), however, by laser-light sources and frequency modulation detection techniques (10–11). When a tunable diode laser is modulated using a radio frequency oscillator, the detection bandwidth can be shifted to higher frequency where intensity fluctuations, inherent to low frequency measurements, are minimized. 

Figure 2: Frequency modulation signals from moisture absorption. The peak-to-peak amplitude of each spectrum is proportional to moisture concentration. The relative humidity is noted to the right of the 0% and 100% scan. 			

The frequency modulation spectroscopy measurement is conducted by frequency modulating a diode laser by superimposing a radio frequency oscillation, Ω, onto the diode injection current. The spectral output of a frequency modulated diode laser (see Figure 1) consists of a carrier frequency ω

  ± Ω. When the laser is scanned through the wavelength of moisture absorbance, the amount of light absorption, which is proportional to the concentration of the water molecules, is "written" into the side band frequencies by recording the difference in absorption between the two sidebands. The differential absorption information is recovered with phase-sensitive detection techniques. The demodulated absorption line shape is shown in Figure 2. The amount of light absorbed is directly proportional to the water concentration in the gas phase. The gaseous water density 

 is related to the peak-to-peak signal amplitude Δ

 by Beer's law, which for a weakly absorbing molecule is given by: 

 is the change in intensity of light after passing through the container (W/cm

 ), Γ is the full-width at half maximum of the absorption signal (cm

				 is the incident laser intensity (W/cm

 is the integrated absorption cross section (cm), and 

 is the container diameter (cm). The measured density in a sample vial is referenced to a standard and displayed as a concentration in percent. The frequency modulated diode laser output is converted to an amplitude modulation after passing through a sample that absorbs at a particular wavelength. The amplitude modulation is detected at radio frequencies and is proportional to moisture concentration.  

Preparation of relative humidity standard.

 We prepared various saturated salt solutions from USP water and ACS-certified salts (Fisher Scientific, Fair Lawn, NJ) for use as relative humidity standards. To use the samples in a headspace moisture analyzer, the saturated salt solutions were placed in an optically transparent sample vial—comprising a glass vial, a rubber stopper, and an aluminum crimp top—typically used for parenteral pharmaceutical formulations. These relative humidity standards were used for instrument calibration and for examining the performance of the headspace moisture analyzer. 

 Pharmaceutical samples included placebo tablets (comprising microcrystalline cellulose, lactose, and magnesium stearate), a gelatin-encapsulated dry product (comprising proprietary drug, sucrose, microcrystalline cellulose, hydroxypropyl cellulose, and sodium lauryl sulfate), a lyophilized product (comprising proprietary drug, gelatin, mannitol, and citric acid), desiccant (silica gel, Süd-Chemie, Munich, Germany), and a liquid-filled hard gelatin capsule (proprietary drug and lipids). To pre-equilibrate the samples at various relative humidity levels, they were stored in weigh boats (single layer) either in a variable temperature and relative humidity stability chamber (Lunaire, Williamsport, PA) or a desiccator with saturated salt solutions for 4–10 days and then sealed into the same vial–stopper–crimp top vial used to prepare the relative humidity standards. 

 A Lighthouse Instruments FMS 1400 headspace moisture analyzer (Lighthouse Instruments, Charlottesville, VA) was used for the frequency modulation studies. The optical head where the sample container is presented (accessible from the top) is continuously purged with nitrogen to eliminate any room air moisture from the measurement region before the experiments. Containers are inserted to the sample holder from the top of the instrument and the measured moisture concentration result is sent to the computer as well as the instrument's front-panel display. The sample holder is jacketed, thus the temperature of the sample holder and the sample can be controlled (see Figure 3). 

Figure 3: Schematic of measuring cell for Lighthouse Instruments FMS 1400 device.			

Typical operation of the instrument was conducted in the following sequence: The sample holder was heated or cooled to the desired measurement temperature. Next, the instrument was calibrated at the measurement temperature using a known activity solution. The water activity reading was graphically monitored in real time to ensure that the vial headspace had reached equilibrium with the sample at the measurement temperature. After the instrument was calibrated, a similar procedure was followed for the actual measurement. Each time the temperature or container was changed or the instrument was shut down, a new calibration was performed.  

Instruments used for comparison in the study.

 A capacitive sensor (Rotronics Hygrolab III, Rotronic Instrument Corp., Huntington, NY) and a chilled-mirror dew-point analyzer (Aqualab 3TE, Decagon Devices Inc., Pullman, WA) also were evaluated for comparison. For studies conducted with the Rotronics Hygrolab III instrument, samples were placed in the measurement chamber that had been previously placed and thermally equilibrated in an circulated-air oven at the desired temperature. The sensor then was placed on top of the measurement chamber and clamped into place. Following equilibration in the sample chamber headspace, the sensor measured the relative humidity at the temperature of interest.  

For the Aqualab 3TE measurements, samples were placed in the measurement chamber that was previously thermally equilibrated at the desired temperature. The samples were allowed to equilibrate, and the relative humidities over the samples were measured.  

Development and application of FMS for water activity measurements.

 The results illustrated the development and application of the FMS 1400 for water activity measurements in pharmaceutically relevant samples. Data taken from several real examples demonstrated the wide applicability of the moisture activity method for pharmaceutical samples.  

We conducted studies on pharmaceutically relevant samples using a FMS sample holder at temperature settings of 25 and 40 °C (see Tables I and II). Measurements were taken on vials containing 10 tablets or capsules, a lyophilized product sample, and a single desiccant. The samples were pre-equilibrated as described previously at 10–84% RH. Tables I and II list the manner of headspace-moisture conditioning. Following this conditioning step, FMS measurements were conducted on the samples at 25 and 40 °C. Various numbers of samples (

) were prepared for repeat measurements, and the average expected %RH was determined.  

In all cases, the difference between the measured RH and the expected RH was within the measurement error expected from the combined sources of sample preparation and instrument bias. The percent relative standard deviation (% RSD) for all measurements also was calculated, and as the data show, the % RSD values for replicate measurements were generally low, ranging from 1–9%. Bias, reported in %RH, also was low and varied from -1 to 1.5 in %RH units. These data demonstrate the capability of the FMS 1400 instrument to measure moisture levels in the headspace over pharmaceutical samples.  

The nondestructive examination of moisture levels in a closed container allows for several applications of the FMS 1400 instrument, including the evaluation of the effect of stopper drying on headspace moisture levels for moisture-sensitive lyophilized products, moisture-vapor transmission measurements of pharmaceutical blister packaging (13), seal integrity testing (14), and the real-time determination of equilibration rates of multicomponent pharmaceutical systems (

 tablets and desiccant within a sealed package.)  

We conducted an experiment to monitor the equilibration of a multicomponent system in real time (see Figure 4). Ten 800-mg tablets of a proprietary product (pre-equilibrated at 25 °C and 60% RH) were sealed in a vial with 2 g of desiccant. The internal RH in the vial was monitored over time using the FMS 1400 instrument, and the results of three replicate experiments were reported (see Figure 4). The multicomponent desiccant–tablet system equilibrated in less than one day, and moisture levels in the package were brought from ~60% RH to a steady-state level of 10% RH. This example illustrates the value of the nondestructive nature of the FMS 1400 analyzer measurement in that it allows for real-time data generation and may improve understanding about the interaction of moisture in various multicomponent systems. 

Figure 4: Results of using the Lighthouse FMS 1400 instrument for monitoring moisture redistribution in a two-component system.			

Comparison with traditional headspace moisture measurement techniques.

 A series of experiments was designed and conducted for comparing the FMS 1400 instrument to traditional chilled-mirror and capacitance-sensor techniques. Three areas were identified as most important for this comparison:  									

the measurement of moisture activity in pre-equilibrated tablets to establish a baseline performance of methods for the prevailing sample type in a pharmaceutical setting; 

moisture retention within the instrument as measured as the bias introduced when a dry sample is preceded by a wet sample;

sample measurement chamber leak rate as determined by measurement of change in moisture activity per hour of a wet pre-equilibrated desiccant sample. 

Figure 5a: Technique accuracy and linearity at 25 Â°C. Plot shows observed relative humidity versus expected relative humidity at a 25 Â°C conditioning temperature.						

One of the fundamental differences between the FMS approach and the other two traditional approaches under examination in the study is that the FMS system uses individual sample vials for each sample that are sealed tightly. The other two approaches use a single sample chamber for all samples. 

Figure 5b: Technique accuracy and linearity at 40 Â°C. Plot shows observed relative humidity versus expected relative humidity at a 40 Â°C conditioning temperature.			

Comparison of pharmaceutical sample headspace-moisture measurements.

 Placebo tablets were pre-equilibrated in stability chambers at two RH levels and three temperatures, for a total of six sample treatment conditions: 25 °C and 20% RH; 25°C and 85% RH; 30°C and 35% RH; 30°C and 65% RH; 40°C and 20% RH; and 40°C and 75% RH. Two replicate measurements were performed on 10 tablets using all three headspace-moisture measurement approaches. Headspace-moisture measurements were acquired at 25, 30, and 40 °C, with tablets pre-equilibrated at the same respective temperatures. Although the FMS 1400 and chilled-mirror techniques both have 

 temperature control over the sample, the capacitive-sensor technique does not and thus the whole apparatus was housed in an oven for temperature control. The capacitive-sensor technique has two measurement settings that render it possible to perform measurements in a relatively "quick" predictive mode or a "standard" mode. Both modes were evaluated in the study (see Table III). A comparison of the biases obtained shows that traditional headspace-moisture approaches and the FMS 1400 instrument all measure headspace moisture with the same degree of accuracy. 			

Measurement bias resulting from instrument moisture retention.  

 The study  evaluated the bias introduced when a dry sample is introduced following measurement on a relatively wet sample. Although we expected the bias for the FMS instrument to be nonexistent because an individual sealed sample vial is used for each sample, bias could be a significant issue for the traditional approaches in which all samples must pass through a single sample measurement chamber.  

Figure 6a: Plot shows percent relative standard deviation versus mean relative humidity for samples at 25 Â°C.

In this experiment, placebo tablets were pre-equilibrated at two humidity levels (at 25 °C and 20% RH and at 25 °C and 85% RH) for the generation of "dry" and "wet" samples for examination. Measurements first were performed on the dry sample, followed by measurement of the more wet sample, and followed again by a measurement on a fresh dry sample. Ten tablets were used for each measurement, and three replicate measurements were conducted. 

Figure 6b: Plot shows percent relative standard deviation versus mean relative humidity for samples at 40 Â°C.			

The capacitive-sensor technique measurements were conducted in the "quick" mode. Table IV shows the average of the three replicate measurements for each instrument type. On the basis of the average readings obtained following measurement on the final dry sample, the chilled-mirror technique may have a significant moisture retention, and the capacitive-sensor technique may have a moderate moisture retention (likely within the measurement error). Very low bias was observed when the FMS 1400 instrument was used, which is entirely consistent with sealed sample vials being used for the measurements. Thus, the FMS 1400 instrument demonstrated that the potential for bias introduction 

Sampling vial and measurement chamber leak rate.

 The seal integrity of the sampling vial or measurement chamber for each technique also was evaluated. In this experiment, a 1-g desiccant canister was pre-equilibrated at 40 °C and 75% RH, and headspace moisture over the desiccant canister was monitored over a 4–8-h period at 40 °C. The change in headspace moisture levels over the time course of the measurement then were translated into an effective leak rate on the basis of the desiccant's quantity and moisture-carrying capacity. The moisture leak rates obtained for the various instruments show that the FMS 1400 instrument (<0.05 mg/h) was superior to the other traditional headspace moisture techniques (Rotronics: 0.5 mg/h; Aqualab: 6 mg/h) in terms of the potential for erroneous data generation resulting from moisture egress or ingress during measurement. The FMS 1400 instrument enables long-term measurements to be made, whereas with the other traditional techniques, it will be difficult to obtain reliable data over long time scales without introducing significant error. 

	Table I: Frequency modulation spectroscopy measurements at 25 Â°C.			

Summary comparison of the various techniques.

 Table V summarizes key attributes of the three headspace-moisture measurement techniques. Comparison points include analysis time, instrument expense, and critical performance variables. As with any instrument comparison, the optimum technique depends on the intended application and the authors' comments should not be taken as an endorsement for any of the instruments.  

All three techniques offer comparable analysis times (except in the Rotronic Hygrolab "quick" mode), although they vary greatly in cost (the FMS 1400 instrument is nearly 10 times more expensive than the capacitive-sensor technique and the chilled-mirror technique). In terms of availability, the FMS 1400 instrument is a rather new approach with the greatest research potential of the three, but availability for the instrument may be somewhat limited. 

	Table II: Frequency modulation spectroscopy measurements at 40 Â°C.			

The FMS 1400 allows the water activity over lyophilized cake samples to be measured directly in a nondestructive manner. As discussed previously, the principal advantage of the FMS 1400 system is the lack of bias introduction that is associated with a sample-vial measuring system, which provides an advantage over using a single measurement chamber for all samples. As the table shows, the FMS 1400 instrument also can be used at a wider range of temperatures than the traditional capacitive-sensor and chilled-mirror techniques. Finally, the FMS 1400 sample vial is not susceptible to the leaking observed with the other two approaches.   

Evaluation of the FMS 1400 instrument performance.

 Because frequency modulation spectroscopy is a relatively new technique, various operational parameters were evaluated to ensure that the instrument could perform as required for routine investigations and was not limited in use to only research studies. Thus, the suitability of the instrument was evaluated in terms of method specificity, accuracy–linearity, and measurement precision and range. 

Frequency modulation spectroscopy is a nondestructive technology for determining the water activity of pharmaceutical samples. This article discusses the various pharmaceutical applications of frequency modulation spectroscopy, offers comparisons with various traditional water activity measurement techniques, and presents an assessment of various instrument performance elements.

Frequency Modulation Spectroscopy

The rapid and accurate determination of headspace oxygen in various pharmaceutical packaging forms poses unique analytical challenges.