ANALYST, JULY 1993, VOL. 118 747 Application of Supercritical Fluid Extraction in the Pharmaceutical Industry: Supercritical Fluid Extraction of Megestrol Acetate From a Tablet Matrix John R. Dean and John Lowdon Department of Chemical and Life Sciences, University of Northumbria at Newcastle, Ellison Building, Newcastle upon Tyne, UK NE I 8ST The factors that influence the extraction of megestrol acetate from a tablet formulation are discussed. A comparison of the Hewlett-Packard HP7680A and a Jasco supercritical fluid extraction (SFE) system is made. Under similar conditions the HP7680A provides a complete recovery of analyte whereas the Jasco system is prone t o analyte loss after depressurization. It is proposed that different SFE methods be compared using the number of cell volumes swept as the criterion of importance.A direct comparison of SFE with a United States Pharmacopeia (USP) monograph method for the analysis of megestrol acetate from tablet formulations has been investigated. Overall, the USP method provided more precise and accurate determinations for the determination of megestrol acetate from a tablet matrix. Keywords: Supercritical fluid extraction; pharmaceutical analysis; megestrol acetate; tablet matrix Hormones are produced by groups of cells (endrocrine glands) which regulate the various life functions in an organism. The steroid hormones form an important group of hormones of which there are five major classes: 1 progestogens, androgens, estrogens, glucocorticoids and mineralocorticoids. At present, both the number and the amounts of artificial steroid hormones used in therapy greatly exceed those of natural hormones used previously from purified animal extracts, as the strength and specificity of the synthetic hormones often exceed that shown by the naturally occurring analogues.' An important hormone involved in the female menstrual cycle is progesterone-1 It is secreted by the corpus luteum in thc ovary and prepares the lining of the uterus for the reception of the ovum and maintains the lining, preventing ovulation after fertilization.The primary usc of the proges- togens is in the treatment of menstrual irregularities and other gynaecological diseases to maintain endangered pregnancies owing to thcir ovulation inhibitory potency. One such synthetically active progesterone compound commonly avail- able as a prescription medicine in tablet formulation is megestrol acetate (3,20-dioxopregna-4,6-dien-17-yl acetate) (Fig.1). Steroid hormones are used in various dosage forms in therapy and are prepared as formulations in tablets, oily or aqueous suspension injectables, ointments and creams. 1 The most convenient means of extracting steroid hormones from tablets is to treat them with a solvent such that the resulting extract can be used directly in the assay method chosen, e.g., extractions with ethanol or methanol, and the test solution can be obtained directly by filtration or centrifugation of the mixture. However, where binding of the active to polar excipients occurs, e.g., corticosteroids with lactose, the extraction loses its effectiveness. In such instances, in order to reduce the adsorption losses and increase selectivity of the extraction, a two-phase extraction is often used: one of the solvents, e.g., water, dissolves the lactose, thus allowing complete extraction by the other solvent which is immiscible with water, e.g., chloroform or diethyl ether.The two-phase extraction is time consuming and often undissolved excipient gives rise to emulsions during the extraction step. In addition to the type and amount of active ingredient, the manner of preparation of the tablet also has to be considered as this can influence the extractability of the steroid. Supercritical fluid extraction (SFE) is a technique that exploits the properties of a substance in a fluid state at temperatures and pressures near the critical point.Of particular relevance to the solvating power of a supercritical fluid, usually carbon dioxide, are the following parameters:2 temperature, density, time of extraction, primary extraction fluid, the method of introduction and choice of modifiers, sample particle size and mode of sample accumulation. Carbon dioxide has so far been the most widely used supercritical fluid because of its low critical temperature (31.1 "C), modcrate critical pressure 17.38 x 106 Pa (73.8 bar)], and high purity at relatively low cost.*J However, the major limitation of carbon dioxide is its inability to extract polar analytes at typical working pressures [2.4 X 1074.8 X 107 Pa (350Ck7000 psi)]. The extraction of polar analytes requires the addition of modifiers (co-solvents) to the carbon dioxide.These co-solvents, e.g., methanol or acetonitrile, can be introduced via a second pump.4 The potential for SFE as a sample preparation technique to extract steroid compounds"." from tablet/excipient formula- tions selectively is currently of interest in the pharmaceutical industry. This paper evaluates the use of SFE to extract megestrol acetate from tablet formulations and suspensions. Initial work indicated that megcstrol acetate was a suitable steroid for investigation not only because of its solubility in supercritical carbon dioxide2 but also because of its solubility in methanol. The latter provided ease of quantification using utlraviolet-visible (UV/VIS) spectrophotometry . Finally, SFE is compared with an official United States Pharmacopeia (USP) method.Instrumentation Two SFE systems were used. The first was a Hewlett-Packard HP7680A computer driven system with total software control and the sccond a Jasco SFE system with dual reciprocating pumps allowing addition of modifier and carbon dioxide. The HP7680A SFE consists of a twin piston pump which allows liquid carbon dioxide (SFC Grade, Air Products, Sunderland, UK) to be pumped at flow rates between 1 and 4 ml min-1 and at prcssures up to 3.65 X lo7 Pa (365 bar). The sample vessel ( I ml capacity) is a stainless-steel tube with PEEK caps fitted with 10 pm frits at either end. The vessel is inserted into a sample well which is raised under computer control to form a high-pressure seal. Extraction can be performed in either static or dynamic mode.In the latter mode carbon dioxide is continuously flowing through the vessel whereas the static mode allows the sample to 'soak' in carbon dioxide. In practice a combination of static and dynamic extraction has748 0 vMe ANALYST, JULY 1993. VOL. 118 ~~~~ Table 1 Typical operating conditions for SFE Instrument Me Fig. 1 Structural formula of megestrol acetate been found to be the most effective to achieve maximum recovery.6 The system pressure is controlled by an electronic- ally actuated variable restrictor. The variable restrictor allows independent control over the flow and pressure of the system. On depressurization the extracted components are collected onto chromatographic support material while the carbon dioxide is vented to waste.In this study Hypcrsil ODS (30 pm) was employed for the collection of the extracted analyte. Finally, analytes are eluted from the support material by selecting an appropriate solvent, i. e . , methanol. The Jasco SFE system utilizes two reciprocating pumps, a master pump (Jasco 880-PU) fitted with a cooling jacket on the pump head to maintain the liquid state of the carbon dioxide and a second pump for the addition of organic modifier. The extraction cell (1.5 ml capacity) is manually wrenched into the thermostatic- ally controlled oven (Jasco 860-CO). Using the dynamic mode, fresh carbon dioxide is continuously swept through the sample cell prior to depressurization. In-line monitoring of the analyte is possible via a UVNIS spectrophotometer (Jasco UV979).Depressurization is achieved using an oscillating variable restrictor (back-pressure regulator, Jasco 880-81). The extracted analyte was collected in a spring-loaded boiling tube or trap into which was placed a few millilitres of solvent (methanol). This collection device was later modified owing to initial problems with anal yte collection. This involved extend- ing the collector into the collection tube by means of a short piece of plastic poly(viny1 chloride) (PVC) tubing over which was placed a small plastic PVC disc. The disc was positioned at the top of the plastic tubing to prevent loss of analyte as an aerosol and encourage condensation of the analyte within the collection tube. Typical operating conditions for both SFE instruments are shown in Table 1.Megestrol acetate standard was supplied by the Standards and Validation Department at Glaxo Manufacturing Services (Barnard Castle, Co. Durham, UK). This material has a certified purity of 99.5% m/m. Megestrol acetate tablets ('Megace') were supplied as 40 and 160 mg formulations by Bristol-Myers Oncology Division (Bristol-M yers Pharmaceut- icals, Langley, Slough, UK). Tablets are the only formulation in which megestrol acetate is currently available. The extracts were quantified at 288 nm using a Uvikon 860 spectrometer. A calibration plot was constructed by serial dilutions of a solution of megestrol acetate in methanol over an appropriate calibration range. Simple lincar regression was applied to the corresponding absorbance and concentration values and a line of best fit determined using a computer package (Statgraphics, Mercia Software, Birmingham, UK).Various support materials were assessed for their suitability as adsorbents, for megestrol acetate in methanol, prior to extraction from the sample vessel. These included: glass beads, 150-200 mesh, 250 GMX; Chromosorb W AW, 100-120 mesh; and molecular sieve, 100-120 mesh 5A, all supplied by Phase Separations (Clwyd, UK). Celite, 80-100 mesh, was supplied by BDH (Poole, Dorset, UK). USP Method for Megestrol Acetate Tablets7 Megestrol acetate tablets were assayed according to the following procedure. Five tablets of each formulation were Parameter HP7680A COX only Fluid delivery- Density/g ml-1 0.65 Pressurehar (kPa) 149 (14 900) Flow rate/ml min-1 2.0 Temperature/"C 55 Equilibration time/min 2.0 Extraction timdmin 5.0 Cell volume/ml 1.5 Cell volumes swept 9.4 Extruction- Depressurizution- Analyte collection ODS column Collectiordwash solvent MeOH * NA = not applicable to this system.Jasco C 0 2 + modifier (if required) 0.66 2.0 151 (15 100) 55 NA* 3.0 1 .0 8.4 Tube McOH individually weighed and placed into calibrated flasks (50 ml capacity for the 40 mg tablets and 200 ml capacity for the 160 mg tablets). To each flask was added 1 ml of water to disintegrate the tablets. This was followed by methanol added to 75% of the fill volume. The flasks were then shaken for 20 min, diluted to volume with methanol, shaken and finally fiItered through a 0.45 pm Acrodisc. Then, 3 ml of filtrate were diluted to 200 ml with methanol and the absorbance recorded in 1 cm cells against a methanol blank at 288 nm.The concentration of megestrol acetate in solution was determined by comparison with the absorbance obtained from a working curve produced using a standard solution of megestrol acetate. Results and Discussion Previous work from this group on steroids has indicated that the main factors that influence extraction are the density, temperature, time and the flow rate of carbon dioxide.6 Using an experimental design approach to investigate the effect of each of the variables and their associated interactions on the recovery of steroids the main influence on the extraction efficiency was determined to be the density of the supercritical carbon dioxide.h With this in mind, compromise extraction conditions, typical of those previously found to give a high recovery of analyte, were selected (Table 1) .6 Recovery of Analyte From Support Materials A 50 p1 aliquot of a standard solution of megestrol acetate (1.208 mg ml-1) in methanol was placed onto a series of support materials using a microsyringe. The solvent was allowed to evaporate to dryness in a current of warm air prior t6 SFE using the HP7680A system and the conditions outlined in Table 1.Care was taken to ensure sufficient time was allowed for the solvent to evaporate to prevent enhanced recovery of analyte due to modification of the supercritical carbon dioxide. Five support materials were chosen to assess the recovery of megestrol acetate (60.4 pg). The five support materials chosen were: a 1 cm glass tube (4 mm i.d.); glass beads (3 g), molecular sieve (0.4 g), Celite (0.2 g) and Chromosorb W AW (0.25 g).The amount of each support required was experimentally determined to be sufficient to adsorb the 50 p1 volume without being present in excess. The results of this recovery exercise are shown in Table 2. With the exception of the molecular sieve data the mean recovery from the other support materials, i . e . , 14 determinations, is 97.4% with a relative standard deviation (RSD) of 6.4%. This is evidence that supercritical carbon dioxide was able to desorbANALYST, JULY 1993, VOL. 118 749 the analyte residue from each of the support materials successfully with the exception of the molecular sieve. The nature of molecular sieves, i .e . , synthetic alkali metal alumi- nosilicates, with regular channels and pores within a lattice structure, provides ideal adsorption surfaces for both the carbon dioxide and analyte to be retained, and hence a longer extraction time may be required to allow the analyte to diffuse out of the matrix. The low recovery of megestrol acetate demonstrates the unsuitability of this support and further work was therefore unnecessary. Celite was selected for further studies. As outlined above, the variable with the maximum effect on the recovery of steroids was determined to be the density of supercritical carbon dioxide. Further investigations were undertaken in order to assess the recovery of megestrol acetate from the Celite support material. The density for extraction was altered by changing the pressure of the system while maintaining a constant temperature.Four densities were selected to investigate the influence of supercritical carbon dioxide density on the extraction of megestrol acetate. The densities selected were: 0.16; 0.43; 0.66; and 0.81 g ml-1. The effect of density on the extraction recovery of megestrol acetate is shown in Table 3. These data and all subscquent experimentation were obtained on the Jasco SFE system. Comparison of the recovery data at a carbon dioxide density of 0.66 g ml-1 allows a direct comparison of the two SFE instruments. It is clearly seen (Tables 2 and 3) that the recovery of megestrol acetate from Celite was substantially reduced using the Jasco system. Every attempt was made to compare extraction conditions between the systems directly but obviously because of equipment restraints (e.g., extrac- tion cell volumes differ) this was not entirely possible.However, as reported previously6 an important feature to consider in comparing data from laboratories and in this instance manufacturer's instrumentation is the number of cell volumes swept. The number of cell volumes swept can be determined using the following equation:h Table 2 Recovery of megestrol acetate from solid support matrices Number of RSD Support material determinations Recovery (%)* (%> Glass tube 5 90.6 (93.2,85.5,92.4, 3.5 92.4,89.5) Glass beads 3 103.4(100.6,105.6,104.0) 2.5 Molecular sieve 3 67.4 (67.5.71.8,62.8) 6.7 Celite 3 103.0(104.3, lOl.l,103.5) 1.6 Chromosorb W AW 3 97.0 (96.9,99.3,94.8) 2.3 * Mean (individual recoveries).Table 3 Effect of density on the recovery of megestrol acetate Number of RSD Density/g ml- 1 determinations Recovcry (%)* (Yo 1 0.16 4 0 0 0.43 4 68.5 (6S.4,67.4.70.0,71.0) 3.7 0.66 4 69.4(68.4,69.3.68.7,71.3) 1.9 * Mean (individual recoveries). Cell volumes swept = (flow rate of C 0 2 x time of extraction)/ volume of cell We have previously recommended that the number of cell volumes swept be at least 4.6 In this instance the number of cell volumes swept was determined to be 9.4 and 8.4 for the HP768OA and Jasco system, respectively. No recovery of megestrol acetate was obtained from the lowest density (below the critical pressure of carbon dioxide at 5.5 "C), thus demonstrating that megestrol acetate is not soluble in gaseous carbon dioxide.Increasing the density of supercritical carbon dioxide at 5.5 "C increases the recovery of megestrol acetate from the Celite support. Assay of Megestrol Acetate Tablets Using the USP Monograph7 Each of the tablet formulations (40 and 160 mg active ingredient) was assayed according to the USP monograph7 for megestrol acetate tablets. The method was adopted to assay content uniformity in the tablets using five tablets of each formulation. The results are shown in Table 4. The assay values are in good agreement with the claimed dosage. The mg per tablet values demonstrate greater variability than the mgg-1 values. This is expected because tablet masses are more difficult to control in the manufacturing process, whereas the mg g-1 values should remain constant if the tablet mix before pressing is homogeneous.Extraction From Tablet Matrix Using Supercritical Carbon Dioxide Analyte recovery from a tablet formulation depends not only on the extraction conditions used but also on physical properties, e.g., particle size, of the matrix. In addition to the physical properties, the extraction process from a tablet matrix involves several steps, including analyte diffusion and the overcoming of adsorption forces associated with the matrix. In order to aid the extraction process from a tablet formulation all tablets were ground to a powder form. The need for grinding was demonstrated by the poor recovery obtained using supercritical carbon dioxide when extracting directly from a tablet (3%).However, the process of grinding can itself lead in some instances to separation of analyte and excipients owing to particle size differences. A 160 mg tablet was first weighed, then ground to a powder. From this, six 20 mg fractions of the sample were removed and extracted over increasingly longer time periods at supercritical carbon dioxide conditions of 55 "C, 0.81 g ml-1 [2.47 x 107 Pa (3579psi)l and a flow rate of liquid carbon dioxide of 2 ml min-1. The results shown in Table 5 indicate that an extraction time of 20 min is required to achieve the maximum recovery of 70.4%. However, it must be assumed that loss of analyte occurs throughout the extraction procedure; this is probably due to loss of analyte from the collection device. Table 4 USP method for megestrol acetate tablets RSDt RSDi Tablet Tablet mass*/g Assay value*/per tablet (Yo 1 Assay value*/mg g-1 (Yo) Nominal 40 mg formulation 0.4036 (O.4O66,0.3972,0.407Oq 40.47 (40.78,39.84,40.63 ~ 0.90 100.28 (100.30, I00.30,99.83, 0.41 0.4OS0, 0.4020) 40.52,40.57) 100.05,100.93) Nominal 160 mg formulation 0.5570 (O.SS66,0.S600, 0.5635, 163.76 (162.93,166.27,167.1 I, 1.81 293.99 (292.72,296.92,296.56, 0.93 * Mean (individual determinations).+ n = 5 . 0.5502 ~ 0.5547) 159.79,162.72) 290.42,293.35)750 ANALYST, JULY 1993, VOL. 118 Extraction From Tablet Matrix Using Modified Supercritical Carbon Dioxide For example, at SO "C the methanokarbon dioxide contains two phases if the pressure is <9S bar (9500 kPa). Modifier The inability of supercritical carbon dioxide to extract compounds that are polar has been highlighted by several workers.2.3 However, the solvating ability of supercritical carbon dioxide can be increased by the addition of modifiers or co-solvents.Conditions necessary to maintain supercritical conditions in the presence of modifiers are well known and recent work4 has highlighted the requirement to ensure that only one phase is present at the selected extraction conditions. Table 5 Effect of extraction time on the recovery of megestrol acetate from a ground tablet using supercritical carbon dioxide Mass of sub-sample/ Time/min mg 5 20.00 10 22.20 15 19.99 20 20.00 25 22.00 30 22.00 Mass of mcgestrol acetate in sub-sample*/ mg 5.88 6.53 5.88 5.88 6.46 6.46 Mass of megestrol acetate recovered/ mg 3.19 4.06 3.61 4.14 4.04 3.50 ' Using mean assay value of 293.99 mg g-l.Recovery 54.3 62.2 61.4 70.4 62.5 54.2 ("/. 1 (10%; methanol) can easily be introduced using the Jasco system via a second reciprocating pump. A similar experiment to that described above was repeated using a ground 160 mg tablet and the results are shown in Table 6. The reported recoveries are significantly higher in the presence of modifier than those reported without modifier (Table 5 ) . Also, maxi- mum recovery (92.8%) was obtained after 20min. These results demonstrate the necessity to add modifier as an aid to SFE, in the presence of a matrix, even for analytes that are known to be soluble in supercritical carbon dioxide. I t is concluded that the presence of a modifier is required to aid diffusion/desorption of the analyte from its matrix.Effect of Sample Mass on Recovery of Megestrol Acetate From a Tablet Using Modified Supercritical Carbon Dioxide Using the optimum conditions previously determined for the SFE of megestrol acetate from a ground tablet matrix, i.e., 0.81 g ml-1, 55 "C and 10% methanol (as modifier) the effect of sample mass was investigated. Five 160 mg tablets were ground to powder form and sub-sampled to give masses of 10, 15, 20, 25, 40 and 50mg. The results, shown in Table 7 , indicate that extraction is independent of sample mass. Table 6 Effect of extraction time on the recovery of megestrol acetate from a ground tablet using methanol-modified supercritical carbon dioxide Mass of Mass of megestrol megestrol Mass of acetate in acetate sub-sample/ sub-sample*/ recovered/ Recovery Time/min mg mg mg (Yo 1 5 20.40 6.00 4.68 78.0 10 21 .OO 6.17 5.38 87.2 1s 20.80 6.11 5.54 90.7 20 23.20 6.82 6.33 92.8 2.5 20.60 6.06 4.95 81.7 30 21.20 6.23 4.91 78.8 * Using mean assay value of 293.99 mg g-'.Table Assay Using Modified Supercritical Carbon Dioxide Each tablet formulation (40 and 160 mg active ingredient) was assayed using modified supercritical carbon dioxide at a density of 0.81 g 1171-1, 55 "C and with 10%" methanol added. The assay was carried out using five tablets of each formula- tion ground to a fine powder in a mortar with a pestle and sub-sampled. The results using modified supercritical carbon dioxide are shown in Table 8. A comparison of SFE with the USP method is shown in Table 9.Conclusions It is clear that SFE introduced some degree of variability into the procedure studied; this will need to be addressed if SFE is Table 7 Effect of sample mass on the recovery of megestrol acetate from a ground tablet using methanol-modified supercritical carbon dioxide Mass of tablet sub-sample/mg 10.50 14.90 23.20 30.10 41 .OO 50. 00 Mass of megestrol acetate in sub-sample*/mg 3.09 4.38 6.82 8.85 12.05 14.70 Mass of megestrol acetate recovered/mg Recovery (YO) 3.23 104.5 4.22 96.3 6.33 92.8 9.08 102.6 11.84 98.3 14.87 101.2 * Using mean assay value of 293.99 mg g-l Table 9 Comparison of USP monograph and SFE assay values USP SFE mg mg mg g-1 per tablet mg g-1 per tablet 40 mg Tublets- Mean 100.3 40.5 98.0 39.5 RSD* (%) 0.4 0.9 4.3 4.9 160 mg Tablets- Mean 294.0 153.8 303.1 169.4 RSD* (Yo) 0.9 1.8 3.0 3.1 * n = 5 .Table 8 Tablet assay results using modified supercritical carbon dioxide Tablet RSDl RSD' Tablet mass'/g Assay value*/per tablet (% 1 Assay valuc*/mg g- (Yo 1 Nominal 40 mg formulation 0.4030 (0.3985,0.043,0.4020, 39.50 (37.76,41.30,37.29, 4.9 98.00(94.74,102.15,92.77. 4.3 0.4065,0.4035) 41.43.39.71) 101.92.98.41) Nominal 160 mg formulation 0.5588(0.5588,0.5580,0.5593, 169.40(169.69,165.99,175.46, 3.1 303.07(303.66,297.05,313.72, 3.0 * Mean (individual determinations). + n = 5 . 0..5600,0.5578) 173.38,162.50) 309.60,29 1.33)ANALYST, JULY 1993, VOL. 118 75 1 to compete as a sample preparation technique with approved methods in the pharmaceutical industry. However, SFE could well replace approved methods if the extraction and assay occurred in a single step using the in-line UVNIS detector. This would provide a more simple analytical procedure than the USP method and the possibility for automation. The authors acknowledge the assistance of M. Kane in the operation of the SFE instrumentation, Glaxo Manufacturing Services for providing the megestrol acetate standard and Bristol-Myers Ltd. for the Megace tablets. The loan of the HP7680A from Hewlett-Packard (UK) Ltd. is also acknow- ledged. In addition, financial support from ICI plc (Dr.s W. R. Campbell and C. J. Dowle) and Glaxo Manufacturing Services (K. Leiper and Dr R. L. Tranter) is gratefully acknowledged. References 1 Gorog, S . , and Szasz. G. Y., Analysis of Steroid Hormone Drugs, Elsevier, Amsterdam. 1978. 2 Hawthorne, S . B . , Anal. Chem., 1990,62, 633A. 3 Dean, J . R., Applications of Supercritical Fluids in Industrial Analysis. Chapman and Hall, London, 1993. 4 Page, S. H., Sumpter, S. R., and Lee, M. L., J . Microcolumn Srp., 1992, 4, 91. 5 Li, S. F. Y., Ong, C. P. Lee, M. L., and Lee, H. K . , J. Chrornatogr., 1990,515, 515. 6 Kane, M., Dean. J. R., Hitchen, S . M., Dowle, C. J . , and Tranter, R. L., Anal. Chim. Acta, 1993, 271, 83. 7 United States Pharmacopeia, USP XXIIlNutional Formulary XVZZ, US Pharmacopeial Convention, Rockville, MD, 1990, Paper 2f06542H p. 815. Received December 9, 1992 Accepted February 3, I993