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Analysis of the Variance Components in a Pharmaceutical Aerosol Product: Lodoxamide Tromethamine
Analysis of the Variance Components in a Pharmaceutical Aerosol Product: Lodoxamide Tromethamine H. A. HAVEL*’,L. J. BEAUBIEN*, AND P. D. HMUND* Received July 26, 1984, from ‘Control Analytical Research and Development and the *Operations Research and Statistical Services, The Upjohn Accepted for publication May 20, 1985. Company, Kalamazoo, MI 49001. Abstract 0 The contributions of several components to the variance in
lodoxarnide delivery from lodoxarnide tromethamine metered-dose aerosol containers have been estimated. Two aerosol lots, manufactured with mean diameters of 2.3 and 7.2 prn, exhibited approximately equal variances. The variance was apportioned to the following components: container-to-container, 27%; mouthpiece-to-mouthpiece 18%; valve-to-valve, 1 1%; assay, 6%. The largest single contribution to the variance (38%) is attributed to unassignable variations which include within-container variations in the dose; quality improvement efforts should concentrate on this area. Little effort should be expended to minimize the assay, valve delivery, or mouthpiece variation as their contribution to lodoxarnide dose variation is small. Likewise, the bulk drug particle size did not contribute appreciably to within-lot dose variation.
Container
I
iL
m Lodoxmnid$i Suspension
Valve L
\
Mouthpiece
Figure 1-Schematic drawing of the lodoxamide suspension aerosol container.
Metered-dose delivery of pharmaceuticals from aerosol containers has proven to be a clinically effective method for SAS (Statistical Analysis System).11J2 The objective of the the treatment of bronchial disease.’ Despite the clinical analysis was to estimate the components of variance due to effectiveness of aerosols, it is widely recognized that the each of the factors listed. The analysis was also planned so as imprecision of commercial aerosol delivery systems has into evaluate homogeneity of the variance structures between hibited the development of aerosol formulations for nonlots and among containers. The assumptions underlying the asthma indications. However, there appears to be a paucity use of the statistical methods were checked as necessary. of data in the literature on the characterization of aerosol dose variation.24 Metered-valve function has also been the subject of few investigationskg. We have thus undertaken a Experimental Section study of the aerosol dose uniformity and metered-valve Design-A nested factorial experiment13J4 was designed to assess functions of single actuations of lodoxamide tromethamiae the effects of three factors and one covariate on variability in aerosol containers to determine the contributions of several lodoxamide dose delivery. The main effects or factors were bulk components to dose variation. particle size (lot), container, and mouthpiece; the covariate was valve Lodoxamide, NJV’-(2-chloro-5-cyano-m-phenylene)dioxa- function. Lots A and B were selected to represent a wide range of mic acid, was administered as a ditromethamine salt in a bulk particle sizes (2.3-7.2 pm). suspension aerosol formulation (Fig. 1).In characterizing the From each lot, four containers were selected at random and actuated -50 times to eliminate any effects due to sampling at the dose variation of this product, we were particularly interestbeginning of the container. In addition, four mouthpieces were ed in the effect of bulk particle size. Although the optimal chosen at random from a supply of mouthpieces. Four single actuaparticle size range for bioavailability considerations is 0.5tions were taken from each container/mouthpiece combinatioq with 10 pm in diameter,10 we had noticed that aerosol lots with the order of selection randomized to minimize any possible effects on larger bulk particle sizes in this range had more dose time of sampling. This design resulted in 128 paired determinations variation than aerosol lots with smaller particle sizes. To of lodoxamine delivery and valve function. The dependent variable investigate this possible particle size effect, we have studied then was the measured quantity of lodoxamide in each actuation. two lots of lodoxamide tromethamine aerosol, with 2.3-pm The covariate was the measured change in weight of the container (lot A) and 7.2-pm (lot B) geometric volume mean diameters. after each actuation (valve function). FN
kl
Lodoxamide
This investigation was based on a general linear-models approach using the factors and covariate as discussed below. All calculations were done using various procedures from 978 / Journal of Pharmaceutical Sciences Vol. 74, No. 9, September 1985
Bulk particle size would ordinarily be a random factor varying over lots. However, in this experiment the two lots were selected purposely for their known bulk particle sizes. Hence, we have treated the main effect of the lot as a fixed effect. We have also reported, as an aside, the variability due to lots which we would have estimated treating lot as a random effect. Containers were selected at random within each lot, so the main effect of container is considered as being a random factor nested within lots. Mouthpieces were also selected at random from the supply available and so were treated as a random factor crossed with containers nested within lots. Replication was performed a t the containerimouthpiece combination level. Thus, the error term contains variation within containers, assay variation, and all other unassignable sources of variation. 0022-3549/85/0900-0978$0 1.00/0 0 1985, American Pharmaceutical Association
The variability due to the assay was estimated separately through an independent experiment and is included in the final results on this basis. Materials-Micronized lodoxamide tromethamine (The Upjohn Co., Kalamazoo, MI) was >98% pure by HPLC and was manufactured into aerosol containers (Riker Laboratories,St. Paul, MN). All other materials were reagent grade or better, used without further purification. Bulk Particle S i z e T h e geometric volume mean diameter of micronized lodoxamide trometvhamine was determined by an electrozone-sensing method16 using a multichannel particle size analyzer interfaced to a digital minicomputer (model 112 LTSAIADCW Particle Data, Inc., Elmhurst, IL). Lodoxamide Dose Sampling-The lodoxamide content of single actuations through-mouthpiecewas determined using the USP unit spray sampling apparatus16 and HPLC analysis (described below). The aerosol container valves were primed and the mouthpiece (Riker Laboratories, St. Paul, MN) was cleaned before each actuation into the sampling apparatus. About 10 mL of an 8 mM phosphate buffer (pH 7.0) solution and exactly 5.0 mL of internal standard solution (see below) were added to the collection chamber before sampling. With the vacuum on (-25 f 2 standard cubic feet per hour), the aerosol container was actuated once, and the vacuum was turned off after -10 s. About 75 mL of water was then used to rinse the lodoxamide residue in the apparatus into the collection chamber. An aliquot of this solution was injected onto the liquid chromatograph. Liquid Chromatographic Analysis-A modular liquid chromatograph was used, consisting of a single-piston pump (model llOA; Altex Scientific, Inc., Berkeley, CA), automatic sampler (model 8000; Varian Instruments, Palo Alto, CAI, injection valve (model AH-CV6H Pax; Valco Instruments, Houston, TX) equipped with a 25-pL loop, octadecyl silica column (p-Bondapak CIS; Waters Associates, Milford, MA), and detector (model 1203; Laboratory Data Control, Riviera Beach, FL) at 254 nm. The mobile phase was methanol:8 mM phosphate buffer (pH 7.0) (5:95). The mobile phase was filtered through a 0.45-pmfilter (Type FH; Millipore Corp., Bedford, MA), deaerated, and pumped at 1.2 mumin. The internal standard solution was a 22 mM salicylic acid solution in methanol:8 mM phosphate buffer (pH 7.0) (10:90).Under these conditions, the retention times of the lodoxamide and salicylic acid peaks were -7 and 10 min, respectively. Quantification of the amount of lodoxamide in each actuation was performed by comparing the peak area ratios (lodoxamide to the internal standard) in sample and reference standard preparations. Valve Function-Each aerosol container was weighed to the nearest 0.1 mg before and after actuation into the USP unit spray sampling apparatus. The difference (in mg) was recorded as the valve function measurement for that actuation. Assay Variance-Accurately weighed amounts of lodoxamide tromethamine were added to the intake tube of the USP unit spray sampling apparatus and washed into the collection chamber with -75 mL water. Other conditions and analysis were the same as for the lodoxamide dose sampling procedure.
Results and Discussion We considered the following assignable sources of variation within a lot: containers; mouthpieces; valve function; assay variation. When sampling from several lots, the lot is also an assignable source of variation. The residual variation (due to unassignable sources) is termed the “error” source. The estimated components of variation are presented in Table I for sampling within a lot and in Table I1 for sampling among lots. When sampling within a lot, i.e., for a fixed bulk particle size, the largest sources of variability are container-to-container (27%) and error (38%) (Table I). The container-tocontainer variation represents lack of uniformity from container to container, all other things being equal. The error term represents variation from dose-to-dose which cannot be accounted for by container and mouthpiece differences or by known fluctuations in container weight per actuation. A possibly significant factor contributing to the error variance term is the within-container variation due to the
Table I-Estimated Components of Variation Within Lots for Lodoxamide Aerosol
Source
Estimated Variance, PS2
Container-to-container Mouthpiece Valve Assay Assignable sources subtotal Error Total
Percent of Total
116 76 47 27 -
27 18 11 6 -
266 161 427
38 __
62 100
homogeneity of the lodoxamide concentration in the suspension. If drug particles settled in the container before actuation, large variability would be observed. This term also includes an unknown percentage of variation which is due to experimental effects, such as analyst, time-of-day, room temperature, etc. These results suggest that in attempting to achieve better dose uniformity within a lodoxamide aerosol lot, it would be most productive. to address container uniformity rather than container-to-container uniformity, mouthpiece uniformity or valve function. Since the reasons for the lack of uniformity of containers are unknown, it may be possible that the same mechanics which result in within-container variation are causing container-to-container variation. Together these two sources of variation represent 70% of the total observed variation in lodoxamide dose within an aerosol lot. When the samples are taken from more than one lot, the variation due to lot is of the same magnitude (28%) as the error variation term (27%) (Table 11). This suggests that a considerable reduction in the variation of lodoxamide dose delivered might be achieved if more uniformity could be established between lots. The two lots which were investigated had a significant mean dose difference, 216 versus 236 pgldose and, as such, are not representative of the lodoxamide aerosol lots routinely manufactured. Thus, the uniformity of lodoxamide aerosol lots is improved in typical lots. The difference in bulk particle size was the only known and measured difference between the two lots included in this study. However, there may be other measurable differences between the two lots of which we are unaware. Fortunately, the assay variation appears to be insignificant when compared to the other sources of variation considered in this study. The variation due to mouthpieces is significant from a statistical standpoint; however, its relative magnitude when compared to other sources of variation suggests that it is not a particularly important source by itself. Table Il-Estimated Components of Variation Between Lots for Lodoxamide Aerosol
Source
Estimated Variance, CLg2
Lot Container-to-container Mouthpiece Valve Assay Assignable sources subtotal Error Total
169 116 76 47 27
Percent of Total 28 19 13
a
__
435 161
5 73 27 -
596
7 00
Journal of Pharmaceutical Sciences / 979 VoL 74, No. 9, September 1985
could not be allocated to an assignable cause was identified as the “error” component. Because the effect of random sampling from several lots is of interest, an additional analysis included an estimated variance component for this source. The primary variance components were estimated from the ANOVA results given in Table V. Because the design was completely balanced, variance component estimates could be obtained by simple calculations based on the expected mean squares, also given in Table V. This method is the same as the default solution for the balanced case when using option MIVQUE(0) in SAS PROC VARCOMP.12 The estimated component for lots was obtained in a second analysis by specifying it as a random effect using the same SAS procedure. This did not effect the estimates for the other components. Estimation of the Valve Function Component-The percent of previously unexplained variation in the dose delivered which could be attributed to valve function was taken to
There is no standard interpretation for the meaning of the component of variation which we have attributed to the valve function. We have considered it to be the variation in dose delivered attributable to weight variation when all other known sources of variation have been removed. Details of Statistical Analysis-In order to maintain a clear focus on the salient points of the analysis, we will first discuss how the variance components were estimated and then provide brief highlights of other parts of the analysis. The experimental results of the lodoxamide dose assays and valve function measurements for lots A and B which were used in this analysis are provided in Tables I11 and IV, respectively. Estimation of Variance Components-The final analysisof-variance model included the following sources of variation with a lot: containers; mouthpieces; error. The secondary sources of variation, valve function and assay variation, were separated from the residual variation in a subsequent analysis. Finally, that portion of the residual variation which
Table Ill-Lodoxamide Dose and Valve Function Results for Lot A (2.3-pm Bulk Particle Size) Mouthpiece A Mouthpiece B Mouthpiece C Container No.
Valve Function,
Lodoxarnide,
Valve Function,
mg
I4
mg
w
I
182 230 221 234
61.8 71.O 72.9 69.5
243 248 248 234
72.8 72.4 72.3 73.1
II
225 241 244 234
70.6 71 .O 70.7 70.8
245 245 273 240
111
199 250 255 250
61.9 72.3 72.8 73.0
IV
231 222 243 227
72.2 71.6 71.6 71.7
Lodoxamide,
w
Lodoxarnide,
Mouthpiece D
Valve Function,
Lodoxamide,
mg
w
226 236 218 22 1
73.3 73.4 73.3 73.1
250 239 238 236
72.4 73.3 72.7 73.1
71.6 71.7 71.6 70.1
224 215 233 223
70.9 70.9 71.3 71.7
260 260 251 242
71.6 72.1 71.9 71.9
241 246 233 255
72.9 71.1 72.6 73.6
217 249 266 253
72.7 73.3 73.2 73.1
252 271 282 256
72.7 72.6 73.7 73.3
294 233 229 217
71.6 72.1 71.6 72.0
206 202 225 195
71.6 71.7 72.0 72.2
227 224 214 210
72.1 71.8 71.8 72.2
mg
Dose and Valve Function Results for Lot B (7.2-pm Bulk Particle Size)
Table IV-Lodoxamide
Mouthpiece A
Mouthpiece 6
Mouthpiece C
Mouthpiece D
Container
No.
Valve Function,
Lodoxamide,
Valve Function,
CLQ
Function, Valve
ma
Lodoxarnide, I4
I
206 229 208 224
71.9 72.4 72.8 72.6
197 219 232 227
II
203 206 169 217
71.3 71.6 59.8 71.6
Ill
196 196 194 219
IV
218 229 237 242
Valve Functior
Lodoxamide,
Function,
cL9
mg
66.8 71.O 69.8 71.O
183 207 217 201
72.5 71.8 71.3 72.1
216 222 216 230
72.4 72.5 72.5 72.1
218 220 206 202
72.2 70.8 71.7 71.4
197 196 195 203
71.4 71.O 70.9 71.4
223 230 202 198
71.5 71.4 71.6 71.8
72.7 72.3 72.7 73.4
222 223 218 207
73.3 73.8 73.7 73.5
215 206 199 214
73.0 73.5 74.2 73.3
213 214 219 227
73.6 73.6 73.9 74.0
74.5 74.3 74.8 74.8
221 250 233 238
74.9 75.1 75.5 75.7
228 221 215 212
75.2 75.4 75.3 75.0
303 250 228 220
74.8 74.5 74.2 74.3
980 /Journal of Pharmaceutical Sciences Vol. 74, No. 9, September 1985
mg
Lodoxamide, I4
mg
be the R2 between the two variables after adjusting each of them separately for the effects of other model terms. The resulting R2 of 20% was then used to adjust the residual variance from the previous analysis of variance to arrive a t the estimated variance component due to valve function. Estimation of th e Assay Component-In order to determine assay variation, a separate experiment was conducted in which solutions containing known quantities of lodoxamide were analyzed. The amount recovered was expressed as a percent of the known amount, and an RSD of 2.3% was calculated (Table VI). Since the overall mean of the lodoxamide delivered for the variance components study was 226.3 pg, we then estimated the SD of the assay to be 5.2 pg. The resulting assay variance was then subtracted from the residual variance from the previous analysis of variance. Selection of the Final Model-In order to estimate variance components, it was necessary to find a common model which would describe variation i n each of the lots. The analysis of the combined data showed that the interaction terms lot-by-mouthpiece (p = 0.8670) and container-bymouthpiece-within-lot (p = 0.2782) were not significant. Thus, the remaining significant factors of containers, mouthpieces, and lots could be included in the variance components analysis (Tables I and 11). Verification of t h e Assumptions-The assumptions of equal variances among containers and between lots were verified before estimating the components of variation in the lodoxamide dose delivered. The test for equal or homogeneous variances among containers using the Levene: 1:meTable V-Analysis
Source
of Variance Results for Final Model
Degrees of Sum of Mean Variance PR > Fa Freedom Squares Squares Ratio (F)
~~~~~
Lot Container (lot) Mouthpiece Model Error Total (corrected)
1 6 3
12940 12547 8001
12940 2076 2667
10 117
33489 27476
3349 235
127
60965
8.90 11.36
0.0001 0.0001
14.26
0.0001
Tests of Hypothesis for Lot Using Container (Lot) as an Error Term PR > F = 0.0473 F = 6.19
Source Lot Container (lot) Mouthpiece
Expected mean square Var(error) + 16 Var [container (lot)] + Q(lot) Var(error) + 16 Var[container (lot)] Var(error) + 32 Var(mouthpiece)
a Probability of
Table VI-Results
achieving a larger F. of Assay Variance Experiment
Lodoxamine Tromethamine, pg Amount Added
Amount Found
461 435 439 449 438 454 455 466 460 413
48 1 428 436 446 444 454 452 454 454 423
Recovery, % 104.3 98.4 99.3 99.3 101.4 100.0 99.3 97.4 96.7 102.4
Mean SD
99.9 2.3
dian testI3J7 was not significant (p = 0.9227). The test for equal variances between the lots using the F test on the mean square errors from the analysis of variance (with factors containers, mouthpieces, and their interaction) also was not significant (p = 0.7330). The within-container means ranged from 205.3 to 248 pg. The within-container SDs ranged from 10.5 to 22.2. Although a number of apparently detached values (suspected outliers) were found in the sample, no values were excluded from the analysis. This decision reflected our findings that no identifiable cause could be associated with these extreme values, that it was desirable to represent the full range of observed values, and that the assumptions of equal (homogeneous) variances among containers and between lots were not violated. The purpose of this study was to assess sources of variation in the amount of the lodoxamide dose delivered from a metered-dose aerosol container. The results of such a study are useful in directing quality-control efforts, assessing process capability limits, and determining an effective productrelease sampling plan. Based on the relative sizes of the sources of variation, we recommend that effort be made to improve container and lot uniformity for this product as opposed to restricting the acceptable bulk particle size range (except for bioavailability considerations) or improving valve function or mouthpiece construction. If these latter two components had been excessively large, we might have recommended that valve or mouthpiece manufacturing processes be made more uniform or new designs implemented for them. The results of such improvements could be monitored by subsequently repeating the dose variation study. A components of variation study similar to that conducted here might be a useful tool in process validation and in determining process capability limits. Such uses would depend on comparing results for a new process against historical results for similar processes. An important use of the results of our study is in determining effective product-release sampling plans. In order to satisfy USP guidelines for dose uniformity, it is necessary to draw a sample of containers and mouthpieces from each lot and analyze a number of determinations of the lodoxamide dose delivered.ls With additional information on the costs of sampling cans and conducting assays, it is possible to devise a sampling plan which would achieve a certain level of confidence a t a minimum cost.19 We can see a number of circumstances under which it would be desirable to repeat a variance components study on a regular basis. Based on our observation that containers provide a relatively higher source of variability than mouthpieces, we would recommend a modified, hierarchical nested design which places more sampling effort at the container leveLt3J4For new products the design chosen appears ideal, as all assumptions could be readily tested. As a final comment, it would be desirable t o spend some additional time studying methods for estimating the component of variation due to a covariate. While we feel comfortable with the method used here, we can make no claims as to its efficiency or optimality.
References and Notes 1. Moren, F. Znt. J . Pharm. 1981, 8, 1-10, 2. Young,J. G.; Porush, I.; Thiel, C. G.; Cohen, S.; Stimmel, C. H. J . Am. Pharm. Assoc., Sci.Ed. 1960,49, 72-74. 3. Lukovits, F. S.; Browning, R. S.; Pratt, E. L. “Advances in Automated Analysis”; Mediced: Tarrytown, NY, 1973; pp 23-27. Journal of Pharmaceutical Sciences / 981 Vol. 74, No. 9, September 1985
4. Scharmach, R. E.Aerosol Age 1982,27,36-40. 5. Porush, I.; Thiel, C. G.; Young, R. G. J. Am. Pharm. Assoc., Sci. Ed. 1960,49,70-72. 6. Contractor, A. M.; Shangraw, R. F.; Richman, M. D. Drug Cosmet. Znd. 1969,105,44-46,126-129. 7. Contractor, A. M.;Richman, M.D.; Shangraw, R. F. J. Phurm. Sci. 1970,59,1488-1491. 8. Cutie, A.; Barger, J.; Clawons, C.; Dolinsk D.; Feinstein, W.; Gupta, B.; Gruenberg, A,; Sciarra, J. J. P k r m . Sci. 1981, 70, 1085-1087. 9. Yu, C. D.; Jones, R.; Wright, J.; Henesian, M. Drug Deu. Znd. Pharm. 1983,9,473-483. 10. Task Grou? on Lung Dynamics Health Phys. 1966,12,173-207. 11. “SAS Users Guide: Basic”; SAS Institute: Cary, NC, 1982. 12. “SAS User’s Guide: Statistics”; SAS Institute: Cary, NC, 1982.
982 /Journal of Pharmaceutical Sciences Vol. 74, No. 9, September 1985
13. Snedecor, G. W.;Cochran, W. G. “Statistical Methods,” 7th ed.; Iowa State University: Ames, 1980. 14. Anderson, V. L.;McLean, R. A. “Design of Experiments”; Marcel Dekker: New York, 1974. 15. Beaubien, L. J.; Vanderwielen, A. J. J. Pharm. Sci. 1980,69, 651-655. 16. “The United States Pharmacopeia,” 20th rev.; US.Pharmacopeial Convention, Inc.; Rockville, MD, 1979; 937 M.’Technonetrics 17. Conover, W.J.; Johnson, M. E.; Johnson, 1981,23,351-361. 18. For example, see the mono aph for isoproterenol sulfate inhalation aerosol. “The United gates Pharmacopeia,” 20th rev.; U S . Pharmacopeial Convention, Inc.; Rockville, MD, 1979,pp 433434. 19. Haaland, P. D.; Havel, H. A,, unpublished results.
&.
lodoxarnide delivery from lodoxarnide tromethamine metered-dose aerosol containers have been estimated. Two aerosol lots, manufactured with mean diameters of 2.3 and 7.2 prn, exhibited approximately equal variances. The variance was apportioned to the following components: container-to-container, 27%; mouthpiece-to-mouthpiece 18%; valve-to-valve, 1 1%; assay, 6%. The largest single contribution to the variance (38%) is attributed to unassignable variations which include within-container variations in the dose; quality improvement efforts should concentrate on this area. Little effort should be expended to minimize the assay, valve delivery, or mouthpiece variation as their contribution to lodoxarnide dose variation is small. Likewise, the bulk drug particle size did not contribute appreciably to within-lot dose variation.
Container
I
iL
m Lodoxmnid$i Suspension
Valve L
\
Mouthpiece
Figure 1-Schematic drawing of the lodoxamide suspension aerosol container.
Metered-dose delivery of pharmaceuticals from aerosol containers has proven to be a clinically effective method for SAS (Statistical Analysis System).11J2 The objective of the the treatment of bronchial disease.’ Despite the clinical analysis was to estimate the components of variance due to effectiveness of aerosols, it is widely recognized that the each of the factors listed. The analysis was also planned so as imprecision of commercial aerosol delivery systems has into evaluate homogeneity of the variance structures between hibited the development of aerosol formulations for nonlots and among containers. The assumptions underlying the asthma indications. However, there appears to be a paucity use of the statistical methods were checked as necessary. of data in the literature on the characterization of aerosol dose variation.24 Metered-valve function has also been the subject of few investigationskg. We have thus undertaken a Experimental Section study of the aerosol dose uniformity and metered-valve Design-A nested factorial experiment13J4 was designed to assess functions of single actuations of lodoxamide tromethamiae the effects of three factors and one covariate on variability in aerosol containers to determine the contributions of several lodoxamide dose delivery. The main effects or factors were bulk components to dose variation. particle size (lot), container, and mouthpiece; the covariate was valve Lodoxamide, NJV’-(2-chloro-5-cyano-m-phenylene)dioxa- function. Lots A and B were selected to represent a wide range of mic acid, was administered as a ditromethamine salt in a bulk particle sizes (2.3-7.2 pm). suspension aerosol formulation (Fig. 1).In characterizing the From each lot, four containers were selected at random and actuated -50 times to eliminate any effects due to sampling at the dose variation of this product, we were particularly interestbeginning of the container. In addition, four mouthpieces were ed in the effect of bulk particle size. Although the optimal chosen at random from a supply of mouthpieces. Four single actuaparticle size range for bioavailability considerations is 0.5tions were taken from each container/mouthpiece combinatioq with 10 pm in diameter,10 we had noticed that aerosol lots with the order of selection randomized to minimize any possible effects on larger bulk particle sizes in this range had more dose time of sampling. This design resulted in 128 paired determinations variation than aerosol lots with smaller particle sizes. To of lodoxamine delivery and valve function. The dependent variable investigate this possible particle size effect, we have studied then was the measured quantity of lodoxamide in each actuation. two lots of lodoxamide tromethamine aerosol, with 2.3-pm The covariate was the measured change in weight of the container (lot A) and 7.2-pm (lot B) geometric volume mean diameters. after each actuation (valve function). FN
kl
Lodoxamide
This investigation was based on a general linear-models approach using the factors and covariate as discussed below. All calculations were done using various procedures from 978 / Journal of Pharmaceutical Sciences Vol. 74, No. 9, September 1985
Bulk particle size would ordinarily be a random factor varying over lots. However, in this experiment the two lots were selected purposely for their known bulk particle sizes. Hence, we have treated the main effect of the lot as a fixed effect. We have also reported, as an aside, the variability due to lots which we would have estimated treating lot as a random effect. Containers were selected at random within each lot, so the main effect of container is considered as being a random factor nested within lots. Mouthpieces were also selected at random from the supply available and so were treated as a random factor crossed with containers nested within lots. Replication was performed a t the containerimouthpiece combination level. Thus, the error term contains variation within containers, assay variation, and all other unassignable sources of variation. 0022-3549/85/0900-0978$0 1.00/0 0 1985, American Pharmaceutical Association
The variability due to the assay was estimated separately through an independent experiment and is included in the final results on this basis. Materials-Micronized lodoxamide tromethamine (The Upjohn Co., Kalamazoo, MI) was >98% pure by HPLC and was manufactured into aerosol containers (Riker Laboratories,St. Paul, MN). All other materials were reagent grade or better, used without further purification. Bulk Particle S i z e T h e geometric volume mean diameter of micronized lodoxamide trometvhamine was determined by an electrozone-sensing method16 using a multichannel particle size analyzer interfaced to a digital minicomputer (model 112 LTSAIADCW Particle Data, Inc., Elmhurst, IL). Lodoxamide Dose Sampling-The lodoxamide content of single actuations through-mouthpiecewas determined using the USP unit spray sampling apparatus16 and HPLC analysis (described below). The aerosol container valves were primed and the mouthpiece (Riker Laboratories, St. Paul, MN) was cleaned before each actuation into the sampling apparatus. About 10 mL of an 8 mM phosphate buffer (pH 7.0) solution and exactly 5.0 mL of internal standard solution (see below) were added to the collection chamber before sampling. With the vacuum on (-25 f 2 standard cubic feet per hour), the aerosol container was actuated once, and the vacuum was turned off after -10 s. About 75 mL of water was then used to rinse the lodoxamide residue in the apparatus into the collection chamber. An aliquot of this solution was injected onto the liquid chromatograph. Liquid Chromatographic Analysis-A modular liquid chromatograph was used, consisting of a single-piston pump (model llOA; Altex Scientific, Inc., Berkeley, CA), automatic sampler (model 8000; Varian Instruments, Palo Alto, CAI, injection valve (model AH-CV6H Pax; Valco Instruments, Houston, TX) equipped with a 25-pL loop, octadecyl silica column (p-Bondapak CIS; Waters Associates, Milford, MA), and detector (model 1203; Laboratory Data Control, Riviera Beach, FL) at 254 nm. The mobile phase was methanol:8 mM phosphate buffer (pH 7.0) (5:95). The mobile phase was filtered through a 0.45-pmfilter (Type FH; Millipore Corp., Bedford, MA), deaerated, and pumped at 1.2 mumin. The internal standard solution was a 22 mM salicylic acid solution in methanol:8 mM phosphate buffer (pH 7.0) (10:90).Under these conditions, the retention times of the lodoxamide and salicylic acid peaks were -7 and 10 min, respectively. Quantification of the amount of lodoxamide in each actuation was performed by comparing the peak area ratios (lodoxamide to the internal standard) in sample and reference standard preparations. Valve Function-Each aerosol container was weighed to the nearest 0.1 mg before and after actuation into the USP unit spray sampling apparatus. The difference (in mg) was recorded as the valve function measurement for that actuation. Assay Variance-Accurately weighed amounts of lodoxamide tromethamine were added to the intake tube of the USP unit spray sampling apparatus and washed into the collection chamber with -75 mL water. Other conditions and analysis were the same as for the lodoxamide dose sampling procedure.
Results and Discussion We considered the following assignable sources of variation within a lot: containers; mouthpieces; valve function; assay variation. When sampling from several lots, the lot is also an assignable source of variation. The residual variation (due to unassignable sources) is termed the “error” source. The estimated components of variation are presented in Table I for sampling within a lot and in Table I1 for sampling among lots. When sampling within a lot, i.e., for a fixed bulk particle size, the largest sources of variability are container-to-container (27%) and error (38%) (Table I). The container-tocontainer variation represents lack of uniformity from container to container, all other things being equal. The error term represents variation from dose-to-dose which cannot be accounted for by container and mouthpiece differences or by known fluctuations in container weight per actuation. A possibly significant factor contributing to the error variance term is the within-container variation due to the
Table I-Estimated Components of Variation Within Lots for Lodoxamide Aerosol
Source
Estimated Variance, PS2
Container-to-container Mouthpiece Valve Assay Assignable sources subtotal Error Total
Percent of Total
116 76 47 27 -
27 18 11 6 -
266 161 427
38 __
62 100
homogeneity of the lodoxamide concentration in the suspension. If drug particles settled in the container before actuation, large variability would be observed. This term also includes an unknown percentage of variation which is due to experimental effects, such as analyst, time-of-day, room temperature, etc. These results suggest that in attempting to achieve better dose uniformity within a lodoxamide aerosol lot, it would be most productive. to address container uniformity rather than container-to-container uniformity, mouthpiece uniformity or valve function. Since the reasons for the lack of uniformity of containers are unknown, it may be possible that the same mechanics which result in within-container variation are causing container-to-container variation. Together these two sources of variation represent 70% of the total observed variation in lodoxamide dose within an aerosol lot. When the samples are taken from more than one lot, the variation due to lot is of the same magnitude (28%) as the error variation term (27%) (Table 11). This suggests that a considerable reduction in the variation of lodoxamide dose delivered might be achieved if more uniformity could be established between lots. The two lots which were investigated had a significant mean dose difference, 216 versus 236 pgldose and, as such, are not representative of the lodoxamide aerosol lots routinely manufactured. Thus, the uniformity of lodoxamide aerosol lots is improved in typical lots. The difference in bulk particle size was the only known and measured difference between the two lots included in this study. However, there may be other measurable differences between the two lots of which we are unaware. Fortunately, the assay variation appears to be insignificant when compared to the other sources of variation considered in this study. The variation due to mouthpieces is significant from a statistical standpoint; however, its relative magnitude when compared to other sources of variation suggests that it is not a particularly important source by itself. Table Il-Estimated Components of Variation Between Lots for Lodoxamide Aerosol
Source
Estimated Variance, CLg2
Lot Container-to-container Mouthpiece Valve Assay Assignable sources subtotal Error Total
169 116 76 47 27
Percent of Total 28 19 13
a
__
435 161
5 73 27 -
596
7 00
Journal of Pharmaceutical Sciences / 979 VoL 74, No. 9, September 1985
could not be allocated to an assignable cause was identified as the “error” component. Because the effect of random sampling from several lots is of interest, an additional analysis included an estimated variance component for this source. The primary variance components were estimated from the ANOVA results given in Table V. Because the design was completely balanced, variance component estimates could be obtained by simple calculations based on the expected mean squares, also given in Table V. This method is the same as the default solution for the balanced case when using option MIVQUE(0) in SAS PROC VARCOMP.12 The estimated component for lots was obtained in a second analysis by specifying it as a random effect using the same SAS procedure. This did not effect the estimates for the other components. Estimation of the Valve Function Component-The percent of previously unexplained variation in the dose delivered which could be attributed to valve function was taken to
There is no standard interpretation for the meaning of the component of variation which we have attributed to the valve function. We have considered it to be the variation in dose delivered attributable to weight variation when all other known sources of variation have been removed. Details of Statistical Analysis-In order to maintain a clear focus on the salient points of the analysis, we will first discuss how the variance components were estimated and then provide brief highlights of other parts of the analysis. The experimental results of the lodoxamide dose assays and valve function measurements for lots A and B which were used in this analysis are provided in Tables I11 and IV, respectively. Estimation of Variance Components-The final analysisof-variance model included the following sources of variation with a lot: containers; mouthpieces; error. The secondary sources of variation, valve function and assay variation, were separated from the residual variation in a subsequent analysis. Finally, that portion of the residual variation which
Table Ill-Lodoxamide Dose and Valve Function Results for Lot A (2.3-pm Bulk Particle Size) Mouthpiece A Mouthpiece B Mouthpiece C Container No.
Valve Function,
Lodoxarnide,
Valve Function,
mg
I4
mg
w
I
182 230 221 234
61.8 71.O 72.9 69.5
243 248 248 234
72.8 72.4 72.3 73.1
II
225 241 244 234
70.6 71 .O 70.7 70.8
245 245 273 240
111
199 250 255 250
61.9 72.3 72.8 73.0
IV
231 222 243 227
72.2 71.6 71.6 71.7
Lodoxamide,
w
Lodoxarnide,
Mouthpiece D
Valve Function,
Lodoxamide,
mg
w
226 236 218 22 1
73.3 73.4 73.3 73.1
250 239 238 236
72.4 73.3 72.7 73.1
71.6 71.7 71.6 70.1
224 215 233 223
70.9 70.9 71.3 71.7
260 260 251 242
71.6 72.1 71.9 71.9
241 246 233 255
72.9 71.1 72.6 73.6
217 249 266 253
72.7 73.3 73.2 73.1
252 271 282 256
72.7 72.6 73.7 73.3
294 233 229 217
71.6 72.1 71.6 72.0
206 202 225 195
71.6 71.7 72.0 72.2
227 224 214 210
72.1 71.8 71.8 72.2
mg
Dose and Valve Function Results for Lot B (7.2-pm Bulk Particle Size)
Table IV-Lodoxamide
Mouthpiece A
Mouthpiece 6
Mouthpiece C
Mouthpiece D
Container
No.
Valve Function,
Lodoxamide,
Valve Function,
CLQ
Function, Valve
ma
Lodoxarnide, I4
I
206 229 208 224
71.9 72.4 72.8 72.6
197 219 232 227
II
203 206 169 217
71.3 71.6 59.8 71.6
Ill
196 196 194 219
IV
218 229 237 242
Valve Functior
Lodoxamide,
Function,
cL9
mg
66.8 71.O 69.8 71.O
183 207 217 201
72.5 71.8 71.3 72.1
216 222 216 230
72.4 72.5 72.5 72.1
218 220 206 202
72.2 70.8 71.7 71.4
197 196 195 203
71.4 71.O 70.9 71.4
223 230 202 198
71.5 71.4 71.6 71.8
72.7 72.3 72.7 73.4
222 223 218 207
73.3 73.8 73.7 73.5
215 206 199 214
73.0 73.5 74.2 73.3
213 214 219 227
73.6 73.6 73.9 74.0
74.5 74.3 74.8 74.8
221 250 233 238
74.9 75.1 75.5 75.7
228 221 215 212
75.2 75.4 75.3 75.0
303 250 228 220
74.8 74.5 74.2 74.3
980 /Journal of Pharmaceutical Sciences Vol. 74, No. 9, September 1985
mg
Lodoxamide, I4
mg
be the R2 between the two variables after adjusting each of them separately for the effects of other model terms. The resulting R2 of 20% was then used to adjust the residual variance from the previous analysis of variance to arrive a t the estimated variance component due to valve function. Estimation of th e Assay Component-In order to determine assay variation, a separate experiment was conducted in which solutions containing known quantities of lodoxamide were analyzed. The amount recovered was expressed as a percent of the known amount, and an RSD of 2.3% was calculated (Table VI). Since the overall mean of the lodoxamide delivered for the variance components study was 226.3 pg, we then estimated the SD of the assay to be 5.2 pg. The resulting assay variance was then subtracted from the residual variance from the previous analysis of variance. Selection of the Final Model-In order to estimate variance components, it was necessary to find a common model which would describe variation i n each of the lots. The analysis of the combined data showed that the interaction terms lot-by-mouthpiece (p = 0.8670) and container-bymouthpiece-within-lot (p = 0.2782) were not significant. Thus, the remaining significant factors of containers, mouthpieces, and lots could be included in the variance components analysis (Tables I and 11). Verification of t h e Assumptions-The assumptions of equal variances among containers and between lots were verified before estimating the components of variation in the lodoxamide dose delivered. The test for equal or homogeneous variances among containers using the Levene: 1:meTable V-Analysis
Source
of Variance Results for Final Model
Degrees of Sum of Mean Variance PR > Fa Freedom Squares Squares Ratio (F)
~~~~~
Lot Container (lot) Mouthpiece Model Error Total (corrected)
1 6 3
12940 12547 8001
12940 2076 2667
10 117
33489 27476
3349 235
127
60965
8.90 11.36
0.0001 0.0001
14.26
0.0001
Tests of Hypothesis for Lot Using Container (Lot) as an Error Term PR > F = 0.0473 F = 6.19
Source Lot Container (lot) Mouthpiece
Expected mean square Var(error) + 16 Var [container (lot)] + Q(lot) Var(error) + 16 Var[container (lot)] Var(error) + 32 Var(mouthpiece)
a Probability of
Table VI-Results
achieving a larger F. of Assay Variance Experiment
Lodoxamine Tromethamine, pg Amount Added
Amount Found
461 435 439 449 438 454 455 466 460 413
48 1 428 436 446 444 454 452 454 454 423
Recovery, % 104.3 98.4 99.3 99.3 101.4 100.0 99.3 97.4 96.7 102.4
Mean SD
99.9 2.3
dian testI3J7 was not significant (p = 0.9227). The test for equal variances between the lots using the F test on the mean square errors from the analysis of variance (with factors containers, mouthpieces, and their interaction) also was not significant (p = 0.7330). The within-container means ranged from 205.3 to 248 pg. The within-container SDs ranged from 10.5 to 22.2. Although a number of apparently detached values (suspected outliers) were found in the sample, no values were excluded from the analysis. This decision reflected our findings that no identifiable cause could be associated with these extreme values, that it was desirable to represent the full range of observed values, and that the assumptions of equal (homogeneous) variances among containers and between lots were not violated. The purpose of this study was to assess sources of variation in the amount of the lodoxamide dose delivered from a metered-dose aerosol container. The results of such a study are useful in directing quality-control efforts, assessing process capability limits, and determining an effective productrelease sampling plan. Based on the relative sizes of the sources of variation, we recommend that effort be made to improve container and lot uniformity for this product as opposed to restricting the acceptable bulk particle size range (except for bioavailability considerations) or improving valve function or mouthpiece construction. If these latter two components had been excessively large, we might have recommended that valve or mouthpiece manufacturing processes be made more uniform or new designs implemented for them. The results of such improvements could be monitored by subsequently repeating the dose variation study. A components of variation study similar to that conducted here might be a useful tool in process validation and in determining process capability limits. Such uses would depend on comparing results for a new process against historical results for similar processes. An important use of the results of our study is in determining effective product-release sampling plans. In order to satisfy USP guidelines for dose uniformity, it is necessary to draw a sample of containers and mouthpieces from each lot and analyze a number of determinations of the lodoxamide dose delivered.ls With additional information on the costs of sampling cans and conducting assays, it is possible to devise a sampling plan which would achieve a certain level of confidence a t a minimum cost.19 We can see a number of circumstances under which it would be desirable to repeat a variance components study on a regular basis. Based on our observation that containers provide a relatively higher source of variability than mouthpieces, we would recommend a modified, hierarchical nested design which places more sampling effort at the container leveLt3J4For new products the design chosen appears ideal, as all assumptions could be readily tested. As a final comment, it would be desirable t o spend some additional time studying methods for estimating the component of variation due to a covariate. While we feel comfortable with the method used here, we can make no claims as to its efficiency or optimality.
References and Notes 1. Moren, F. Znt. J . Pharm. 1981, 8, 1-10, 2. Young,J. G.; Porush, I.; Thiel, C. G.; Cohen, S.; Stimmel, C. H. J . Am. Pharm. Assoc., Sci.Ed. 1960,49, 72-74. 3. Lukovits, F. S.; Browning, R. S.; Pratt, E. L. “Advances in Automated Analysis”; Mediced: Tarrytown, NY, 1973; pp 23-27. Journal of Pharmaceutical Sciences / 981 Vol. 74, No. 9, September 1985
4. Scharmach, R. E.Aerosol Age 1982,27,36-40. 5. Porush, I.; Thiel, C. G.; Young, R. G. J. Am. Pharm. Assoc., Sci. Ed. 1960,49,70-72. 6. Contractor, A. M.; Shangraw, R. F.; Richman, M. D. Drug Cosmet. Znd. 1969,105,44-46,126-129. 7. Contractor, A. M.;Richman, M.D.; Shangraw, R. F. J. Phurm. Sci. 1970,59,1488-1491. 8. Cutie, A.; Barger, J.; Clawons, C.; Dolinsk D.; Feinstein, W.; Gupta, B.; Gruenberg, A,; Sciarra, J. J. P k r m . Sci. 1981, 70, 1085-1087. 9. Yu, C. D.; Jones, R.; Wright, J.; Henesian, M. Drug Deu. Znd. Pharm. 1983,9,473-483. 10. Task Grou? on Lung Dynamics Health Phys. 1966,12,173-207. 11. “SAS Users Guide: Basic”; SAS Institute: Cary, NC, 1982. 12. “SAS User’s Guide: Statistics”; SAS Institute: Cary, NC, 1982.
982 /Journal of Pharmaceutical Sciences Vol. 74, No. 9, September 1985
13. Snedecor, G. W.;Cochran, W. G. “Statistical Methods,” 7th ed.; Iowa State University: Ames, 1980. 14. Anderson, V. L.;McLean, R. A. “Design of Experiments”; Marcel Dekker: New York, 1974. 15. Beaubien, L. J.; Vanderwielen, A. J. J. Pharm. Sci. 1980,69, 651-655. 16. “The United States Pharmacopeia,” 20th rev.; US.Pharmacopeial Convention, Inc.; Rockville, MD, 1979; 937 M.’Technonetrics 17. Conover, W.J.; Johnson, M. E.; Johnson, 1981,23,351-361. 18. For example, see the mono aph for isoproterenol sulfate inhalation aerosol. “The United gates Pharmacopeia,” 20th rev.; U S . Pharmacopeial Convention, Inc.; Rockville, MD, 1979,pp 433434. 19. Haaland, P. D.; Havel, H. A,, unpublished results.
&.