Anal. Chem. 1998, 70, 2762-2765
Quantitative Determination of Bucindolol Concentration in Intact Gel Capsules Using Raman Spectroscopy Thomas M. Niemczyk,* Miriam M. Delgado-Lopez, and Fritz S. Allen
Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131
The ideal quality control method for pharmaceutical products should be capable of rapid nondestructive testing of intact tablets or capsules. Raman spectroscopy using near-infrared excitation is shown to be capable of obtaining useful spectral data directly from drug formulations in gel capsules and from the gel capsules inside blister packs. The Raman data collected from the capsules inside blister packs containing 0-100 mg of the active ingredient (bucindolol), when coupled with multivariate calibration, resulted in a calibration SEP of 3.36 mg. The largest source of error was found to be due to sample inhomogeneity. Even so, the method is shown to have significant potential as a rapid nondestructive quality control method for pharmaceutical samples. Total quality management in the pharmaceutical industry requires that a variety of sample types be completely characterized. Samples include bulk dry substances and excipients, as well as formulations that are made up of mixtures of the two. When packaged for distribution, the samples might be in the form of tablets or gel capsules contained in bottles or blister packs. Current quality control procedures of the final product often take the form of an assay for the active ingredient in a formulation. To carry out these assays, the product is often subjected to crushing (or grinding), dissolution, separation, and finally determination of the active ingredient. The determination step can often be automated, but the sample procedures are time consuming and destructive and thus are only applied to small random samples of capsules or tablets. A method that can rapidly provide good quantitative results directly on tablets or capsules without any sample preparation would be highly desirable. One of the advantages that is often discussed for near-infrared (NIR) spectroscopy is that very little sample preparation is often required.1 Indeed, a number of papers have appeared in the literature that have discussed near-infrared techniques for the analysis of pharmaceutical samples.2-7 Generally, NIR analysis requires only that a sample be crushed and the spectral data can (1) Burns, D. A.; Ciurczak, E. W. Handbook of Near-Infrared Analysis; Dekker: New York, 1992. (2) Kirsch, J. D.; Drennen, J. K. Appl. Spectrosc. Rev. 1995, 30, 139-174. (3) Ciurczak, E. W. Appl. Spectrosc. Rev. 1987, 23, 147-163. (4) Corti, P.; Dreassi, E.; Lonardi, S. Il Farmaco 1993, 48, 3-20. (5) Josefson, M.; Jedvert, I.; Johansson, S.; Langkilde, F. W. Eur. J. Pharm. Sci. 1994, 2, 82-83. (6) Plugge, W.; Van der Vlies, C. J. Pharm. Biomed. Anal. 1992, 10, 797-803.
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be obtained very rapidly; hence, NIR analysis can be carried out rapidly with minimal sample preparation. NIR data are, however, complicated. Nonparametric multivariate analysis or other patternrecognition techniques are required to interpret the data. The combination of nonparametric multivariate analysis and NIR data can be very powerful, as demonstrated by Lodder and Hieftje, who showed the combination could be used to monitor intact tablets,8 detect tampering in gel capsules,9 and detect contamination in drug capsules.10 Aldridge et al. extended these methods to show that NIR and multivariate analysis could be used to discriminate different tablet formulations inside blister packages.11 Recently, NIR and multivariate analysis have been used to determine the salicylic acid content of degraded aspirin tablets12 and the concentration of a vasodilator in tablets.13 Of note in the latter application is that tablet placement in the spectrometer was a significant source of noise in the measurements. Recently it has been suggested that Raman spectroscopy can be used to determine the contents of a tablet or gel capsule.14 Often, the encapsulants used in tablets, or the gel capsule itself, are nearly transparent to the radiation employed in Raman spectrometry. It has been shown that high-quality Raman spectra can be obtained directly from a tablet.14 These data were useful in the determination of the tablet contents, but to date it has not been shown that Raman spectroscopy can be used to make a quantitative determination of the active ingredient in intact formulations. In this note, we discuss the application of nearinfrared Raman spectroscopy to make quantitative determinations of gel capsule contents. Good results are obtained directly from gel capsules, as well as from gel capsules contained in blister packs. EXPERIMENTAL SECTION The bucindolol samples were 500-mg ((10%) formulations in gel capsules, which were supplied by the Veterans Administration (7) MacDonald, B. F.; Prebble, K. A. J. Pharm. Biomed. Anal. 1993, 11, 10771085. (8) Lodder, R. A.; Hieftje, G. M. Appl. Spectrosc. 1988, 42, 556-558. (9) Lodder, R. A.; Selby, M.; Hieftje, G. M. Anal. Chem. 1987, 59, 1921-1930. (10) Lodder, R. A.; Hieftje, G. M. Appl. Spectrosc. 1988, 42, 1500-1512. (11) Aldridge, P. K.; Mushinsky, R. F.; Andino, M. M.; Evans, C. L. Appl. Spectrosc. 1994, 48, 1272-1276. (12) Drennen, J. K.; Lodder, R. A. J. Pharm. Sci. 1990, 79, 622-627. (13) Monfre, S. L.; DeThomas, F. A. Near Infrared Spectroscopy: Bridging the Gap Between Data Analysis and NIR Applications; Hildrum: Chichester, U.K., 1992; p 435. (14) Petty, C. J.; Bugay, D. E.; Findlay, W. P.; Rodriguez, C. Spectroscopy 1996, 11 (5), 41-45. S0003-2700(97)01252-3 CCC: $15.00
© 1998 American Chemical Society Published on Web 05/15/1998
Cooperative Studies Program. The active ingredient in the gel capsules, bucindolol, was present at different nominal levels: 0, 3, 6.25, 12.5, 25, 50, and 100 mg. Assays of capsule contents (15 examples of each concentration) show the capsule contents to be, respectively, 3.03 ( 0.15, 6.27 ( 0.18, 12.21 ( 0.36, 24.46 ( 0.91, 48.35 ( 2.70, and 96.14 ( 2.12 mg, when the capsule content mass is normalized to 500 mg. An excipient was used to make up the remaining mass of the gel capsule content. The gel capsules were clear and supplied in blister packs. The Raman spectral data were collected using a Chromex Raman 2000 spectrometer equipped with a Chromex distally filtered fiber-optic Raman probe. The Raman probe is configured so that scattering is collected from a spot ∼500 µm in diameter when the sample is positioned optimally. The spectrometer was equipped with a 785-nm laser that delivered ∼95 mW to the sample. A 600 lines/mm grating blazed at 1 µm was used in all experiments. This grating results in a reciprocal linear dispersion of 4.5 nm/mm at the CCD camera. The CCD camera was a Photometrics model SDS-9000A, which is 27.6 mm wide. The combination of camera width and reciprocal linear dispersion allows a region of ∼1730 cm-1 to be simultaneously imaged onto the camera. The gel capsules were sampled by setting them into a slot which had been milled into a thin metal plate. The fiber-optic Raman probe was positioned directly below the slot so that it was looking up at the gel capsule. Spectra of the samples in the blister packs were obtained by mounting the blister packs on an XY translation stage. The blister pack was mounted on the bottom of the XY translation stage with the foil side against the stage. The Raman probe was placed below the blister pack and positioned to look up at the samples. The positioning precision of the XY movement was specified to be better than (10 µm. All data processing, including data preprocessing, was performed using the PLSNGR software written at Sandia National Laboratories to operate within the GRAMS environment. All data were processed using the partial least-squares (PLS) algorithm after mean centering both the reference and calibration data. Cross-validation was always employed with either a single sample rotated out or with all examples of a particular formulation rotated out during the calibration process. The predictive ability of the PLS models is reported as the cross-validated standard error of prediction (SEP). When comparing SEPs, significant differences were determined using an F-test at the 90% confidence level. RESULTS AND DISCUSSION Bucindolol is a β blocker vasodilator that has shown potential in the treatment of heart disease.15 Both the bucindolol molecule
Figure 1. Raman spectra obtained from (a) excipient (powder), (b) bucindolol (powder), (c) 50-mg bucindolol formulation (powder), and (d) 50-mg bucindolol formulation (gel capsule). Spectra a-c have been offset for clarity.
spectra with considerable feature overlap in the 400-1500-cm-1 region. This is in contrast to excipients such as cellulose, which is a weak Raman scatterer. Thus, the bucindolol formulations represent a challenging application. Spectra of pure bucindolol, the excipient, a 50-mg formulation (spectrum obtained from the powder), and a 50-mg sample in a gel capsule (spectrum obtained through the gel capsule) are shown in Figure 1. As can be seen, there are features of the bucindolol that are relatively isolated from the excipient bands. These include the C-C aromatic ring mode features in the 1520-1630-cm-1 region and the C-N stretch at 2226 cm-1. Also, note that the spectrum obtained through the gel capsule looks identical to that obtained from the powder. No features due to the gel capsule are apparent. Raman spectra were obtained from gel capsules, two or more of each of the formulations, using both 30- and 90-s integration times. Calibrations were performed focusing on the spectral data between 1500 and 2400 cm-1, the region that produced the best calibrations with powdered samples.16 The 30-s integration data resulted in an SEP of 2.90 (single sample rotation) and 3.63 mg (multiple sample rotation) using only a linear baseline correction. The data obtained with a 90-s integration resulted in an SEP of 2.58 mg (multiple sample rotation) using only a linear baseline correction. The data were also subjected to a Savitsky-Golay smooth. When the 90-s integration data were subjected to a ninepoint Savitsky-Golay smooth, the multiple sample rotation SEP improved to 2.37 mg. PLS calibration results are summarized in Table 1. Cross-validation is often employed when a multivariate calibration model is being developed. Cross-validation aids in the selection of the optimum number of factors used by the calibration model, helps to identify outlier samples in the calibration set, and is used in creating statistical models that can identify outliers when unknown samples are encountered. The PLS calibration results summarized in Table 1 were all obtained using two-factor models, the optimum number of factors determined during the crossvalidation process. Most often single sample rotation is used when the cross-validation process is performed. Care must be taken, even when using cross-validation, that all samples are unique if
and the excipient used in these formulations have rich Raman (15) Eichhorn, E. J. Am. J. Cardiol. 1993, 71, 65C-69C.
(16) Niemczyk, T. M.; Delgado-Lopez, M. M.; Allen, F. S.; Clay J. T.; Arneberg, A. L. Appl. Spectrosc., in press.
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Table 1. Calibration Results Obtained from Bucindolol Gel Capsules no. of integration samples time (s) 25 25 19 19 a
30 30 90 90
data pretreatment LBCa LBC LBC LBC and S-Gb (9 pt)
C-V SEP rotation (mg) single multiple multiple multiple
2.90 3.63 2.58 2.37
R2 0.992 0.984 0.993 0.994
LBC, linear baseline correction. b S-G, Savitsky-Golay smooth.
Figure 3. Raman spectra of 50-mg bucindolol samples (a) obtained from a gel capsule and (b) obtained from a gel capsule within a blister pack. Spectrum a has been offset for clarity. Table 2. Cross-Validated Calibration Results Obtained from Bucindolol Gel Capsules inside Blister Packs Using Multiple Sample Rotation and 60-s Integration
Figure 2. The cross-validated PLS calibration plots obtained for (b) formulations in gel capsules, R 2 ) 0.994, SEP ) 2.37 mg, and (4) formulations in gel capsules within blister packs, R 2 ) 0.986, SEP ) 3.23 mg.
the SEP generated is to be a reliable measure of how well future unknown samples will be predicted by the model. When the calibrations under discussion here were performed, several different capsules containing presumably identical formulations were included in each calibration set of samples. Even though the capsules are different, and even though the sampling noise seems to be dominate (see below), not removing all examples of the same formulation during the cross-validation process leads to a model that has probably overfit the data. Note the significant difference between the SEP obtained from the same set of calibration data using single sample rotation and multiple sample rotation (Table 1). Note also in Table 1 that an increase in the integration time and the use of Savitsky-Golay smoothing improve the SEP of the calibration. This suggests that there are sources of highfrequency noise contributing to the signals being measured. Integration times longer than 90 s led to saturation of the most intense peaks in the Raman spectra. The cross-validation calibration plot obtained using the 90-s integration data and the nine-point Savitsky-Golay smooth is shown in Figure 2. A calibration was also carried out limiting the spectral region to the isolated bucindolol bands. This calibration used the data between 1520-1640 and 2210-2240 cm-1. The SEP based on these data using multiple sample rotation was 2.24 mg, essentially equivalent to the results obtained using the larger spectral range. This corresponds to the results obtained from the powdered samples.16 Figure 3 contains spectra obtained from a 50-mg gel capsule and a 50-mg gel capsule within a blister pack. As can be seen, 2764 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998
no. of samples
data pretreatment
no. of factors
SEP (mg)
R2
37 37 62 62
LBC LBC and S-G (9 pt) LBC LBC and S-G (9 pt)
2 3 2 3
3.23 3.22 3.36 3.41
0.986 0.989 0.987 0.987
the spectra appear identical with the exception of a ∼17% loss in intensity when the data are obtained through the blister pack. Presumably the loss of intensity is due to reflective losses as the radiation, both the excitation beam and the Raman scattering, passes through the curved blister pack wall. Two data sets obtained from samples within blister packs were collected, in both cases using 60-s integration times. The first set contained 37 samples and the second set, collected several months later, contained 62 samples. The spectral data over the 1500-2400cm-1 frequency region were subjected to PLS modeling and the results are summarized in Table 2. As can be seen in Table 2, the use of Savitsky-Golay smoothing allows the model to use one more factor but does not result in improved SEPs. Indeed, an F-test applied to the SEPs shows that they are statistically equivalent. The cross-validation calibration plot produced from the 37-sample set using only linear baseline correction is shown in Figure 2. An experiment to test the reproducibility of this procedure was carried out. A blister pack containing 12.5-mg capsules was mounted on the XY stage. Four different capsules, different locations on the blister pack, were sequentially sampled by moving the XY stage to position the individual capsules above the fiberoptic probe. Sixty-second integrations were used, and allowing for the movement time from position to position, the individual samples were measured 6 times at ∼6-min intervals. The PLS model developed from the entire 62-sample set was then used to predict the amount of bucindolol from these spectral data. The average bucindolol amount and the standard deviation for each of the four blister pack positions are summarized in Table 3. When the data from the four positions are combined, the average
Table 3. Results Obtained from Repeat Sampling from Four Positions of a Blister Pack Containing 12.5-mg Bucindolol Gel Capsules position
av bucindolol amt (mg)
std dev (mg)
1 2 3 4
12.13 14.73 13.20 14.31
1.04 0.98 1.01 1.12
bucindolol amount is 13.59 mg and the standard deviation is 1.42 mg. An experiment was also carried out where repeat samplings of an individual position was performed without moving the XY stage between samplings. Again, 60-s integrations were used with a 60-s delay between repeat samplings. The data resulting from this experiment were again predicted using the PLS calibration model based on the data from the entire 62-sample set. The standard deviation of these repeat determinations was 0.37 mg. CONCLUSION The data presented here show that it is possible to obtain quantitative results using Raman spectroscopy on intact gel capsules, even gel capsules in blister packs. The method is rapid, nondestructive, and relatively precise. The precision of the method is within the (10% variation to which the gel capsules are made up at the higher dosage levels and is sufficient to classify the very lowest dosage levels. The fact that the data can be obtained through the blister packs suggests great potential as a final stage production line monitoring technique. The best SEP obtained directly from a powdered sample was 1.97 mg,16 which is significantly better than that obtained here. There are two differences that might cause the difference in precision. More light, i.e., more signal, was collected from the smoothed surface of the powdered sample relative to that collected through the gel capsule and blister pack materials. The loss of light is, however, not as big an effect as is the sample-to-sample differences seen when the intact capsules are sampled. Noise in the data due to the CCD camera, laser instability, and anything else affecting the signal-to-noise level in an individual data collection, is reflected in the SEP of 0.37 mg obtained in the repeat measurements. As expected, these noise sources are impacted by increased signal averaging (Table 1). The largest source of noise encountered in these determinations is due to problems associated with sampling a heterogeneous
solid sample. The strongest evidence for this statement is in Table 3. The standard deviation of repeat measurements on the same sample(s) was ∼1 mg, and the average determination for supposedly identical samples differed by 2.6 mg. Given the fact that the positioning precision of the XY stage, better than (10 µm, was much better than the spot size, ∼500-µm diameter, the area of the sample measured during the reproducibility study can be considered identical. The heterogeneity of the samples is such that there are significant sample-to-sample differences seen within the area sampled. Another contribution to sampling noise is change within an individual sample caused by vibrations transmitted to the sample from the stepper motor of the XY stage. We believe these changes contribute to the ∼1-mg precision measured when moving the sample, versus the 0.37-mg precision with no movement. The precision of the measurements reported here could be improved by increasing the signal-to-noise ratio (SNR) of the measured data. Longer integration times would improve the SNR, but camera saturation and lower sample throughput must be considered. Longer effective integration times can be achieved by the use of block averaging, but any SNR ratio increase is dependent upon performing the block averaging correctly.17 The use of a CCD camera that produces better SNR would also be of benefit.16 The collection of more scattered radiation would likely improve the measurement, something that could be achieved by use of a higher power laser or more efficient collection optics. The largest noise source is due to the heterogeneity of the powdered sample formulations. Little can be done about the samples if measurements are to be made without removing them from the blister packs. Collection of data from a larger area of the sample than used might average out some of the differences due to the heterogeneity. An irradiated spot the size of half the gel capsule diameter, 3.5 mm, seems a reasonable goal. Whether this is accomplished with a fiber-optic probe or a system of mirrors/lenses, the collection efficiency of scattered radiation will decrease. In a situation such as that discussed here, where sample inhomogeneity is a limitation, this seems like a good tradeoff.
Received for review November 13, 1997. Accepted April 4, 1998. AC971252U (17) Chase, D. B. Pittcon ′98, New Orleans, LA, March 1-5, 1998; Paper 693.
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