for In Situ Analysis of Residual Moisture and Methanol Robert A. Mattes,* Rudolf Schroeder, Vinny Dhopeshwarkar, Robert Kowal, and William Randolph ear-infrared (NIR) spectroscopy is an analytical tech- nique based on absorption measured between the visi- ble and mid-infrared electromagnetic spectral region. Strong fundamental absorption bands of moieties occur in the mid-IR region and frequently require mulls or dilutions to bring the absorbances within the detectors linear range. The over- tone absorptions enable direct measurement without sample preparation because the absorption is relatively weak. Economic fiber-optic cables can be used to measure processes that are lo- cated far from the analyzer by inserting probes into processing equipment. For more than a decade, NIR spectroscopy and fiber-optic probes have been used for real-time process monitoring (1, 2). The turbulent flow of powder granules, however, poses a chal- lenge for high-shear granulator moisture analysis monitoring. For example, the turbulence constantly and randomly changes the apparent sample density in front of the NIR probe window, making accurate analysis impossible. When the sample density change is brought within an acceptable narrow range, NIR analy- sis can be performed by multivariate methods that account for density variance as well as moisture variance. This paper dis- cusses a novel probe design to reduce the powder-sample den- sity variance such that moisture and solvent levels can be ac- curately modeled and predicted. Determining a formulations moisture level is essential for subsequent process capability and product stability. Over- drying wastes energy, ties-up critical facilities, and can damage the formulation because of hydration changes in some active ingredients and excipients (3, 4). Typically, moisture content is monitored off-line with Karl Fischer volumetric titrimetry or at-line with thermogravimet- ric loss-on-drying (LOD) methodology (5, 6). Karl Fischer and LOD analyses usually require the operator to stop the process, use a sample thief to collect a sample, and then analyze the sam- ple while the drying process continues. It is not possible to ob- tain real-time moisture level trends. In addition, Karl Fischer analysis is costly, time-consuming, and uses chemical reagents that require purchase and proper disposal. LOD analysis is not L . B .
B O H L E N A novel probe design can reduce the powder-sample density variance such that moisture and solvent levels can be accurately modeled and predicted in process. The single-pot high-shear granulator (L.B. Bohle, Warminster, PA) used in the study. Robert A. Mattes is an instrumentation scientist at FOSS NIRSystems, Inc., 7703 Montpelier Road, Laurel, MD 20723 tel. 301.680.7251, fax 301.236.0134, rmattes@foss-nirsystems.com. Rudolf Schroeder, PhD, is a pharmaceutical scientist at L.B. Bohle (Ennigerloh, Germany). Vinny Dhopeshwarkar, PhD, is a principal scientist in the Technical Development group, Robert Kowal, RPh, is a manager of technical operations, and William Randolph, PhD, is a senior director of techical operations, all at PSGA. A Division of Ortho-McNeil Pharmaceutical (Titusville, NJ). *To whom all correspondence should be addressed.
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eration (approximately two complete scans of 32 co-added scans per minute) result- ing in 150 spectra per batch. Three test batches and three calibration batches were run. All spectral data collection and analy- ses were performed with Vision TM soft- ware (FOSS NIRSystems). Results and discussion Moisture analysis. Figure 1 shows spectra with no math treatment (raw spectra) of the raw materials used in the granulation. Figure 2 illustrates the second-derivative spectra of the raw materials. The second derivitive is commonly used in NIR spec- troscopy to remove the baseline offset caused by scattering and to en- hance absorbance peaks (11). The second-derivative spectral peaks appear inverted with respect to the raw spectra (12). Figure 3 shows the samples raw spectra collected during the drying cycle. Water absorbs strongly in the 1400 and 1900-nm NIR range, as evidenced by the peaks in those regions. Figure 4 shows an en- larged view of a region used to model the samples moisture and methanol content. Because of the second-derivative math treatment, the moisture content increases in the down- ward direction in this region. The LOD re- sults taken for the raw materials and the gravimetric percent added during granu- lation were used as the initial value for modeling the moisture during the drying cycle. At least four degrees of freedom were modeled in this study: methanol content, water content, temperature variance, and moisture specific because all volatiles con- tribute to the thermogravimetric loss. In addition, monitoring the removal of granulation liquids such as methanol is important for product purity. Typically, methanol level is measured by taking a sample with a sample thief and sending it to the laboratory for high-performance liquid chromatography (HPLC) analysis. HPLC requires long elution times, uses solvents that must be disposed of prop- erly, and requires calibration samples to be run frequently. NIR spectroscopy is a rapid, non- destructive technique that can provide real-time analysis without sample prepa- ration and can store data automatically. NIR spectroscopy is well-suited for the process analytical technology (PAT) ini- tiative proposed by the Food and Drug Administration (710). NIR can moni- tor low levels of residual moisture, alco- hol, and other process constituents to yield better process control and quality man- agement. Experiment A process instrument (FOSS NIRSystems, Laurel, MD) was set up and connected to a fiber-optic probe with a specially de- signed tip, which was installed into a sin- gle-pot high-shear granulator (VMA 70, L.B. Bohle, Warminster, PA). The probe (FOSS NIRSys- tems) has an angled face that diverts the powder sample in a quasilaminar flow over the measurement window. This design main- tains a constant sample den- sity at the window interface. A 24.0-kg charge of 91% lactose anhydrous and 9% starch 1500 was prepared and loaded into the gran- ulator. The granulator blended the dry mixture to homogeneity for 10 min. Water, methanol, and yel- low dye (FD&C #6) were sprayed into the mixture and blended. The yellow dye measured in the parts- per-billion range and was not a source of interference with NIR measurements. NIR spectra were collected continuously during the blending and granulation operations. Samples for LOD analysis were withdrawn at specific inter- vals. Each NIR spectrum consisted of 32 co-added sample and reference scans in the 8002100-nm NIR range. After the moisture and methanol were added and blended, an agglomeration cycle was run for 6 min. The heated jacket was energized, and a vacuum was pulled to 200 mbar to start the drying opera- tion. The drying operation was uniform and gradual over a period of 43 min with an agitator speed of 200 rpm and a nitro- gen purge rate of 1.0 NM 3 /h at standard temperature and pressure. NIR spectra were collected continuously during the en- tire blending, granulation, and drying op- Table I: LOD samples and NIR predictions. MeOH H 2 O% LOD H 2 O% MeOH% Blend Sample LOD% NIR NIR NIR 4 7 1.22 1.23 4 67 10.1 1.73 8.37 8.22 4 104 5.3 1.32 3.98 3.64 4 137 2.10 0.89 1.21 0.32* 4 176 1.10 0.70 0.40 0.75 4 204 1.27 0.53 0.74 0.35 5 206 1.35 1.38 5 315 9.90 1.70 8.20 7.82 5 346 4.95 1.43 3.52 4.44 5 381 1.97 0.97 1.00 0.45 5 410 1.25 0.77 0.48 1.07 5 432 1.07 0.64 0.43 0.49 6 468 1.36 1.32 6 563 9.42 1.75 7.67 7.77 6 604 3.42 1.28 2.14 1.94 6 631 1.90 0.99 0.91 0.71 6 663 1.34 0.76 0.58 0.93 6 693 0.96 0.63 0.33 0.12 * indicates that sample did not pass library qualification For the study, the standard temperature probe was replaced with the angled NIR probe. A novel probe (FOSS NIRSystems) with an angled sample interface. density variance. Figure 5 shows the NIR-predicted samples versus the values calcu- lated from LOD values. The moisture levels with time were not precisely known because the LOD accounts for moisture and methanol losses. For example, Table I shows that blend 4 initially had 1.22% moisture before wet granulation; 0.5% moisture was added dur- ing granulation resulting in 0.53% after drying. This method was re- peated for blends 5 and 6. These values and the linearly interpolated in- termediate values were applied to the spectra ac- quired at corresponding times. A three-factor par- tial least squares (PLS) regression model was de- veloped with the 18 data points in Table I. The second derivative inten- sity and the standard normal variate (SNV) (6) for scatter correction over the 11002100-nm range were used to develop the model with multiple correlation coefficient (R 2 ) value of 0.9541 and a standard error of calibration (SEC) of 0.0966%. The standard error of cross-validation was 0.1835, which is not unreasonable for a small sample set. The spectra from the drying cycle used to develop the pre- diction model were used to develop a library to qualify samples for quantification. This library and the prediction model were then used in a routine analysis function to predict on the aver- age of five consecutive samples. The results are plotted in Fig- ure 6. Some initial samples were not predicted when the aver- age spectrum did not meet library qualification because of high rpm and turbulence during the blending cycle. Although the true values are partly theoretical, the predictions are reasonable even through the granulation cycle (not modeled). Methanol analysis. Figure 4 shows the second-derivative spec- tra of blend 5s analytical region as an example of the spectra used for predicting methanol levels in the lactose anhydrous and starch 1500 blend. To estimate the methanol level the NIR- predicted moisture values were subtracted from the LOD val- ues. A model was made from the resulting 17 adjusted values (see Table I). According to the qualification library, one spec- trum was an outlier and was not used in the model. As shown in Figure 7, a PLS regression model was developed using the second derivative intensity over the 18002100-nm range and resulted in an R 2 value of 0.9823, and an SEC of 0.4427%. The standard error of cross-validation was 0.6224. These samples are not well spaced because of the faster initial methanol removal. The water 2.9136 2.6222 2.3308 2.0395 1.7481 1.4568 1.1654 0.8741 0.5827 0.2914 0.0000 A b s o r b a n c e Wavelength (nm) 8 0 0 9 0 0 1 0 0 0 1 1 0 0 1 2 0 0 1 3 0 0 1 4 0 0 1 5 0 0 1 6 0 0 1 7 0 0 1 8 0 0 1 9 0 0 2 0 0 0 2 1 0 0 Figure 1: Raw spectra of raw materials. 0.7899 0.7108 0.6316 0.5525 0.4734 0.3942 0.3151 0.2360 0.1568 0.0777 -0.0014 A b s o r b a n c e Wavelength (nm) 8 0 0 9 0 0 1 0 0 0 1 1 0 0 1 2 0 0 1 3 0 0 1 4 0 0 1 5 0 0 1 6 0 0 1 7 0 0 1 8 0 0 1 9 0 0 2 0 0 0 2 1 0 0 Figure 3: Raw spectra of drying samples. 0.0120 0.0087 0.0053 0.0020 -0.0014 -0.0047 -0.0081 -0.0115 -0.0148 -0.0182 -0.0215 A b s o r b a n c e Wavelength (nm) 1 8 5 0 1 8 6 1 1 8 7 1 1 8 8 2 1 8 9 3 1 9 0 4 1 9 1 4 1 9 2 5 1 9 3 6 1 9 4 6 1 9 5 7 1 9 6 8 1 9 7 9 1 9 8 9 Increasing H 2 O Figure 4: Second-derivative spectra in the analytical region. 0.2925 0.2309 0.1694 0.1079 0.0464 -0.0151 -0.0766 -0.1382 -0.1997 -0.2612 -0.3227 A b s o r b a n c e Wavelength (nm) 8 0 0 9 0 0 1 0 0 0 1 1 0 0 1 2 0 0 1 3 0 0 1 4 0 0 1 5 0 0 1 6 0 0 1 7 0 0 1 8 0 0 1 9 0 0 2 0 0 0 2 1 0 0 Figure 2: Second-derivative spectra of raw materials. lactose anhydrous starch 1500 methanol water 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 N I R - p r e d i c t e d
( % ) Theoretical moisture (%) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Figure 5: Moisture Calibration set: NIR predicted values versus those calculated from LOD data in Table I. R 2 value is 0.9541 and SEC is 0.0966%. was removed in a quasi-linear function of time. The methanol level, however, was nonlinear, thus leaving less data in the mid- level. To develop a robust model, more data must be taken ini- tially. The use of HPLC as a reference method provides more accurate results. Figure 8 is the process trend chart from routine analysis for the granulation and drying operation. These spectra were ana- lyzed in routine analysis, averaging five spectra for each predic- tion, and tested against a qualification library. The methanol level of some initial samples was not predicted by the routine analy- sis when the average spectrum did not meet library qualification be- cause the granulator and turbulence had a high rpm during the blend- ing cycle. The methanol level reached a minimum and then increased slightly before reaching the final minimum. This effect was most likely caused by the removal of free methanol, the fracturing of granules, and the release of additional interstitial methanol. Again, although the true values are partly theoretical, the predictions are smooth and reasonable throughout the granulation cycle that was not modeled. Conclusion The in situ NIR spectroscopic method predicted water and methanol loss in samples of lactose anhydrous and starch 1500 during the blending, granulation, and drying operations. Although the accu- racy is not known, the trended predictions appear smooth and rea- sonable from known initial and end-point data. The single-pot gran- ulator trial indicates that an NIR process instrument with the novel angled probe can measure a moving sample in turbulent flow when it is placed in the sample medium in a manner that provides suffi- ciently constant sample density. Proper probe placement and design in process equipment is essential for successful implementation of in situ measurements. With HPLC reference data for the methanol and Karl Fischer ref- erence data for the moisture, the accuracy and precision of the model predictions should be improved. It should be possible to model par- ticle size, blend uniformity, and temperature with reasonable preci- sion if proper reference data are provided. Acknowledgments The authors wish to thank Karl Norris and Katherine Bakeev for their review and valued comments on this manuscript. References 1. J.B. Callis, D.L. Illman, and B.K. Kowalski, Process Analytical Chemistry, Anal. Chem. 59 (9), 624A635A (1987). 2. K.A. Bakeev, Near-Infrared Spectroscopy as a Process Analytical Tool: At- Line and On-Line Applications and Implementation Strategies, Spectroscopy 19 (1), 3942 (2004). 3. S.M. Maggard, D.E. Root, and M. Duell, On-line Monitoring of Pharma- ceutical Dryers, J. Process Anal. Chem. 7 (1) (2002). 4. R.M. Leasure and M.K. Gangwer, Near-Infrared Spectroscopy for In-Process Moisture Determination of a Potent Active Pharmaceutical Ingredient, Am. Pharm. Rev. 5 (1), 103109 (2002). 5. E.W. Ciurczak and J.K. Drennen III, Pharmaceutical and Medical Applica- tions of Near-Infrared Spectroscopy (Marcel Dekker, New York, NY, 2002). 6. M.B. Hicks et al., In Situ Moisture Determination of a Cytotoxic Com- pound During Process Optimization, J. Pharm. Sci. 92 (3), 529535 (2003). 7. M.L. Balboni, Process Analytical Technology Concepts and Principles, Pharm. Technol. 27 (10), 5467 (2003). 8. FDA Draft Guidance PATA Framework for Innovative Pharmaceutical Manufacturing and Quality Assurance, August 2003, http://www.fda.gov/cder/OPS/PAT.htm. 9. H. Forcinio, Pharmaceutical Industry Embraces NIR Technology, Spec- troscopy 18 (9), 1624 (2003). 10. R.C. Lyon et al., Exploring Pharmaceutical Applications of Near-Infrared Technology, Am. Pharm. Rev. 6 (3), 6270 (2003). 11. T.C. OHaver and T. Begley, Signal-to-Noise Ratio in Higher-Order De- rivative Spectrometry, Anal. Chem. 53 (12), 18761878 (1981). 12. H. Mark and J. Workman, Jr., Derivatives in Spectroscopy: Part I, The Be- havior of the Derivative, Spectroscopy 18 (4), 3237 (2003). PT 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 M e t h a n o l
( % ) Methanol 5:09:36 5:16:48 5:24:00 5:31:12 5:38:24 5:45:36 5:52:48 6:00:00 6:07:12 6:14:24 6:21:36 1.8 Figure 6: The moisture process trend chart for the granulation and drying operations for blend 4. 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 -1.0 N I R - p r e d i c t e d
m e t h a n o l
( % ) Theoretical methanol (%) -1.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 Figure 7: Methanol Calibration set: NIR predicted values versus those calculated from LOD data in Table I. R 2 value is 0.9823 and SEC is 0.4427%. 8 7 6 5 4 3 2 1 0 M e O H
( % ) Time 5:09:36 5:16:48 5:24:00 5:31:12 5:38:24 5:45:36 5:52:48 6:00:00 6:07:12 6:14:24 6:21:36 9 Figure 8: The methanol process trend chart for the granulation and drying operations for blend 4. Reprinted from PHARMACEUTICAL TECHNOLOGY, September 2004 Printed in U.S.A. 7703 Montpelier Road Laurel, MD 20723 U. S. A. T: 301.680.9600 F: 301.236.0134 E: info@foss-nirsystems.com www.foss.dk