Effect of HPLC Analytical Procedure upon Determining Drug Content in PLGA Microspheres

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Sunju Heo, Honghwa Lee, Minjung Lee and Hongkee Sah

^†

College of Pharmacy, Ewha Womans University, 11-1 Daehyun-dong, Seodaemun-gu, Seoul 120-750, Korea

(Received May 16, 2010·Revised May 31, 2010·Accepted June 1, 2010)

ABSTRACT−The objective of this study was to investigate the effects of sample preparation, HPLC conditions and peak measurement methods upon determining progesterone content of poly-d,l-lactide-co-glycolide microspheres. A series of the microspheres with different formulations was first prepared. To determine their actual drug contents, the microspheres were dissolved in tetrahydrofuran and diluted with various amounts of methanol to precipitate the polymer. After removal of poly- meric precipitates, the filtrates were subject to HPLC analysis under versatile experimental conditions. Interestingly, the composition of a sample solution (e.g., the ratio of methanol to tetrahydrofuran) affected the magnitudes of both peak fronting and peak broadening of progesterone. Its peak became broader and more asymmetrical at lower methanol:tetrahydrofuran ratios. Furthermore, its peak height was influenced by the proportion of tetrahydrofuran in a sample solution. Such problems encountered with tetrahydrofuran were exacerbated when a larger volume of the sample solution was injected onto an analytical column. Under our experimental conditions a peak area measurement provided more accurate and reliable determination of progesterone content in various microspheres than a peak height determination. Optimizing the composition of a sample solution, HPLC chromatographic conditions and peak analysis methods was a prerequisite to an accurate determination of progesterone encapsulated within microspheres.

Key words−Poly-d,l-lactide-co-glycolide, Microspheres, Incorporation Efficiency

Biodegradable poly-^d,l-lactide-^co-glycolide (PLGA) polymers are widely used as delivery systems for various low- molecular-weight compounds, proteins, vaccines, and DNA (Pawar et al., 2004; Moshgeghi and Peyman, 2005).In par- ticular, PLGA microspheres can provide controlled release of drugs over a wide range of delivery durations, when they are administered intramuscularly or subcutaneously into patients.

Commercial PLGA microsphere products include Lupron Depot, Sandostatin LAR Depot, Somatuline LA, Risperdal Consta, Vivitrol, and Parlodel LA. In literature, a number of studies have also been reported with regard to the microencapsulation of contraceptive steroids, such as progesterone and related estrogens, into PLGA microspheres (Beck et al., 1981;

Benoit et al., 1986; Sah, 2000).

The contents of progesterone in various dosage forms are often determined by HPLC systems using the mixture of water and methanol as a mobile phase (Formento et al., 1987; Wu et al., 2000; Pucci et al., 2003). In common practice, a sample solution to be injected to HPLC is recommended to have the same solvent composition like a mobile phase. However, methanol-water mixtures at different volume ratios cannot dis-

solve PLGA microspheres. It is likely that the use of the cosol- vent systems provides incomplete extraction of progesterone entrapped in PLGA microspheres, thereby leading to the underestimation of its microencapsulation efficiency. The use of an organic solvent that dissolves completely PLGA polymers might aid in overcoming such a limitation. Tetrahy- drofuran can be of the choice, since it is a low-viscosity solvent having excellent solvation power on a wide range of natural and synthetic polymers. For example, most gel per- meation chromatography systems shooting for the measurement of polymer molecular weight are operated by using tetrahydrofuran (Mehta et al., 1996; Leamen et al., 2004;

Gaborieau et al., 2008). In addition, tetrahydrofuran is used in the assay of drug products. For instance, the Japanese Phar- macopoeia describes that for the content assay of bufexamac cream, the dosage form is dissolved in tetrahydrofuran and then diluted with a mobile phase before HPLC analysis. Sim- ilarly, a common analytical procedure used to determine drug content of PLGA microspheres first dissolves the drug-loaded PLGA microspheres in organic solvents such as tetrahydrofuran and methylene chloride (Schaefer and Singh, 2001). A known quantity of methanol is then added until the polymer precipitates out of the solution. After removal of the pre- cipitated polymer through filtration, the filtrate is subject to HPLC analysis.

†Corresponding Author :

Tel : +82-2-3277-4367, E-mail : hsah@ewha.ac.kr DOI : 10.4333/KPS.2010.40.3.193

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So far, little attention has been paid to the effects of sample preparation, HPLC chromatographic conditions and peak analysis methods upon the assay accuracy of drug microencapsulation efficiency. In this study it is supposed that the difference between an injection sample solution and a mobile phase might affect the peak shape of progesterone and subsequently its microencapsulation efficiency. Changes in peak properties often lead to poor separation and have undesirable effects on the calculation of peak area and/or height for quantitative analysis. Therefore, this study aimed at scrutinizing the influence of the composition of a sample solution, the type of a mobile phase and peak analysis methods upon determining the drug content of PLGA microspheres. Progesterone was used as a drug to be encapsulated into the microspheres throughout this study.

Experimental

Materials

A poly-^d,l-lactide-^co-glycolide with a lactide:glycolide ratio of 75:25 (inherent viscosity, 0.70 dL/g in CHCl3 at 30^oC) was purchased from Lactel Absorbable Polymers (AL, USA). This polymer was abbreviated as PLGA in text. Polysciences, Inc.

(PA, USA) was the supplier of polyvinyl alcohol (88% hydro- lyzed, MW = 25,000). An ammonia solution (28%), ethyl chloroacetate, and ethyl fluoroacetate were purchased from Junsei Chemical Co., Ltd (Tokyo, Japan). Progesterone was obtained from Sigma-Aldrich (MO, USA). Ammonium acetate was purchased from Daejung Chemicals and Metals Co., Ltd. (Gyonggi-do, Korea). Methanol, acetonitrile, and tetrahydrofuran of HPLC grade were obtained from Burdick &

Jackson (MI, USA).

Preparation of PLGA Microspheres by Ammonolysis- Based Microencapsulation

Progesterone was encapsulated into PLGA microspheres via an ammonolysis-based microencapsulation technique reported elsewhere (Sah and Lee, 2006).Briefly, PLGA (0.25 g) and progesterone (60, 100, 160, 200, or 250 mg) were dissolved in 4 mL of ethyl fluoroacetate. The dispersed phase was emul- sified in 40 mL of a 0.5% aqueous polyvinyl alcohol solution with a magnetic stir plate (800 HPS/VWR Scientific Co.).

After 3 min stirring, 9 mL of the ammonia solution was added to the oil-in-water emulsion, which was stirred for 30 min.

Ammonolysis led to the conversion of water-immiscible ethyl fluoroacetate into water-miscible ethanol and fluoroacetamide.

These events provided the quick transformation of emulsion droplets into PLGA microspheres. After 40 mL of water was

added into the microsphere suspensions, they were passed through a 425^µm sieve, collected by filtration and stirred again in 80 mL of a 0.1% aqueous polyvinyl alcohol solution for 2 h. At this time, the microsphere suspension was stirred at 500 rpm. They were then separated by filtration and dried overnight in a vacuum oven. Microspheres with a given formulation were prepared at least 3 batches, in order to evaluate batch-to-batch reproducibility. Microspheres were also prepared by using ethyl chloroacetate, instead of ethyl fluoroacetate, under the same experimental conditions described earlier.

Scanning Electron Microscopy (SEM)

Scanning electron micrographs of PLGA microspheres were taken for the assessment of their surface and internal morphology. Some microsphere samples were embedded in an epoxy resin and cross-sectioned to expose their internal structure, prior to sputter-coating. The morphology of the microspheres was then observed by a JSM-5200 electron microscope (Jeol Inc.; MA, USA) at 10 kV.

HPLC Systems

Chromatographic measurements were performed on a Shi- madzu model LC-20A series HPLC equipped with a binary pump (LC-20AD), a UV/VIS detector (SPD-20A), and an autosampler (SIL-20A). The system was controlled by the Shi- madzu LC Solution 1.12 software. It was the Luna C18 5 mm column (150×4.6 mm) that was used as an analytical column.

A mixture of methanol and water (80:20, by v/v) was used as a mobile phase at the flow rate of 0.8 mL/min. The elution of progesterone was detected at a wavelength of 254 nm. Another mobile phase used in this study was a mixture of 0.02 M ammonium acetate buffer (pH 4.5) and acetonitrile (44:56, by v/v). Its flow rate was set at 1.0 mL/min, and the eluent containing progesterone was measured at 214 nm. Unless otherwise stated, the injection volume of a sample solution was fixed at 20^µL.

Progesterone Standard and Sample Solutions

Progesterone standard solutions (16.6, 76.1, 133.1, 199.7, and 266^µg/mL) were prepared with methanol. Methanolic progesterone solutions containing different proportions of tetrahydrofuran were also prepared to investigate the effect of tetrahydrofuran upon the peak shape of progesterone. To do so, 5 ~ 3 parts of a methanolic progesterone solution were mixed with 1 ~ 3 parts of tetrahydrofuran, respectively. The final concentration of progesterone in the sample solutions ranged from 76.1 to 133.1 and 199.7^µg/mL.

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Effect of Injection Volume of a Sample Solution upon Progesterone Peak

A methanol-tetrahydrofuran mixture (4:2) was prepared to have a progesterone concentration at 199.7^µg/mL. The injection volume of the sample solution was changed from 10 to 15, 20, 25, 30, 40 and 50^µL. The injection volume effect upon the peak shape of progesterone was then evaluated by use of the asymmetry factor.

Peak Measurement of Progesterone

Quantitative information on progesterone was obtained from both its peak area and peak height from HPLC chromatograms.

An automatic peak height method was used to measure the signals by the detector as the peak height using the Shimadzu LC Solution data processing system. Another method used for measuring peak was an automatic integration method measuring the signals by the detector as the peak area using the Shimadzu data processing system. At the same time, the asymmetric factor was used to quantitatively describe the peak shape of progesterone obtained under various chromatographic conditions. It was calculated from the chromatographic peak by dropping a perpendicular at the peak apex and a horizontal line at 10% of the peak height.

Determination of Progesterone Microencapsulation Efficiency

Microsphere samples, accurately weighed, were dissolved in 4 mL of tetrahydrofuran. One part of the solution was further diluted with 5 parts of methanol, to precipitate the PLGA polymer. The suspension was filtered with a nylon syringe fil- ter (0.45^µm pore size) to remove PLGA precipitates. Aliquots of the filtrate (20^µL) were injected onto the Luna C18 5^µm column. The samples were eluted isocratically with the mobile phase consisting of methanol and water, following the HPLC conditions described earlier. Progesterone peaks in the chromatograms were quantitated by both peak height and peak area measurements. The percentage microencapsulation efficiency of progesterone was calculated as follows:

Microencapsulation efficiency% = 100×

where actual loading is ,

and theoretical loading is

Results and Discussion

Our microencapsulation process made it possible to prepare spherical PLGA microspheres. Fig. 1 shows the external and internal morphology of the microspheres prepared using 0.25 g of PLGA and 160 or 250 mg of progesterone. Their internal morphology is featured with tiny cavities present across the microsphere matrices. An attempt was made to determine progesterone content in the microspheres by dissolving the PLGA microspheres in tetrahydrofuran, diluting with methanol to precipitate out the PLGA polymer and subjecting the sample solution to the HPLC analysis.

Before the measurement of the progesterone content in the microspheres, first investigated was the influence of the composition of a sample solution and the type of a mobile phase upon the peak of progesterone. Fig. 2 shows the peaks of progesterone dissolved in methanolic solutions with different proportions of tetrahydrofuran. In case when the methanol- water mixture was used as a mobile phase, increases in the proportion of tetrahydrofuran in a sample solution led to considerable reductions in the peak height of progesterone (Fig.

2A). It was also observed that the more tetrahydrofuran a sample solution contained, the broader was the peak. As shown in Fig. 2B, substitution of the mobile phase with the ammonium acetate-acetonitrile mixture helped alleviate the peak distortion of progesterone to some extents. However, the replacement still accompanied some changes in the peak properties of progesterone in the presence of tetrahydrofuran.

The deviation degree of progesterone peak from a sym- metric shape was expressed by calculating the asymmetry factor. For a symmetrical peak, the asymmetry factor becomes 1.

On the contrary, its number is less than 1 for a fronting peak, whereas a value greater than 1 is attained with a tailing peak.

In general, asymmetry factors of 0.9 to 1.2 are considered acceptable for test compounds. When progesterone was eluted with the methanol-water mobile phase, the value of the asymmetry factor was dependent upon the proportion of tetrahydrofuran in a sample solution: as the portion of tetrahydrofuran increased, the peak became more asymmetric and broader.

This conclusion was applicable for all sample solutions containing different concentrations of progesterone (Fig. 3A). For example, at methanol:tetrahydrofuran ratios of 6:0 and 5:1, their asymmetry factors were in the range of 1.126±0.010 to 1.008±0.001 (mean±s.d.; n =5). However, their asymmetry factors were less than 0.9 at the volume ratios of 4:2 and 3:3.

These substantiated that tetrahydrofuran triggered the occur- rence of considerable peak fronting. In contrast, the magnitude of peak fronting was less severe when the methanol-water actual loading

theoretical loading wt. of progesterone in microspheres wt. of microspheres taken for analysis

wt. of progesterone used for microencapsulation total wt. or progesterone and PLGA used to prepare microspheres

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mobile phase was switched to the ammonium acetate-acetonitrile one (Fig. 3B). In this case, the asymmetry factors fell between 0.946±0.001 and 1.150±0.002. It is known that peak fronting occurs under a variety of conditions. Some typical causes of peak fronting include column void, column overload, column channeling, analyte-mobile phase/solvent interactions, poor solubility of an analyte in a mobile phase, and its slow

reaction (Forgács et al., 2001; Keunchkarian et al., 2006). Our results demonstrate that differences in the solvent composition between a sample solution and a mobile phase are closely associated with peak fronting.

The consequence of peak distortion caused by tetrahydrofuran was evaluated in the following studies. The measurements of progesterone concentrations were performed on the

Figure 1. SEM micrographs illustrating the surface and internal morphology of PLGA microspheres prepared using 9 mL of ethyl fluoroacetate as a dispersed solvent. The amounts of progesterone used for microencapsulation were (a, b) 160 and (c, d) 200 mg.

Figure 2. HPLC chromatograms of 133.1 µg/mL progesterone solutions having different solvent compositions. The progesterone solutions consisted of methanol and tetrahydrofuran at the ratio of (a) 6:0, (b) 5:1, (c) 4:2, and (d) 3:3, respectively. Two kinds of mobile phases were used: (A) the methanol-water mixture and (B) the ammonium acetate-acetonitrile one.

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progesterone standard solutions with 3 known concentrations (76.1, 133.1, and 199.7^µg/mL). In this experiment progesterone standard solutions were always prepared with methanol, and the methanol-water mixture was used as a mobile phase.

When a peak area measurement was carried out, changes in the composition of a sample solution did not bring about any noticeable inaccuracy in the concentration measurements:

There were close agreements between the progesterone concentrations measured and those of known values. Table I sum- marizes the relevant accuracy and precision data of our assay carried out under different experimental conditions and peak analysis methods. As seen in Fig. 2, the significant cost of peak distortion triggered by tetrahydrofuran was loss in peak height.

Due to reductions in the peak height, progesterone concentrations quantified by a peak height measurement were far

away from their true values. After the methanol-water mobile phase was substituted with the ammonium acetate-acetonitrile one, similar conclusions were drawn (Fig. 4). In this case, however, the level of inaccuracy observed with a peak height measurement was less severe than that observed with the methanol-water mixture.

Generally, the measurement of a peak is carried out to quan- tify a test compound by peak height or peak area methods.

Many analysts prefer a peak area method over a peak height method, since the former is thought to be more robust than is the latter (Issaq and Young, 1977). A peak height measurement is of choice for some occasions (Taylor et al., 2005; Ha et al., 2006). An example is a case that adjacent peaks near the peak of a test compound have a potential of influencing its peak; in this case, a peak height measurement provides smaller error than does a peak area measurement. Our results showed that both peak area and peak height measurements provided acceptable precision and accuracy, only when the methanol:tetrahydrofuran ratio of a sample solution was 6:0. In the presence of tetrahydrofuran, however, the peak height of progesterone decreased to a great extent. Subsequently, a peak height measurement provided false information on progesterone concentrations of the sample solutions. Interestingly,

Figure 3. Dependence of the asymmetry factor upon the composition of a sample solution. Various volume ratios of methanol to tetrahydrofuran were used to prepare progesterone solutions at 3 concentrations. The mobile phases used were (A) the methanol-water mixture and (B) the ammonium acetate-acetonitrile mixture.

Table I. Effects of the Composition of a Sample Solution upon Intraday Precision and Accuracy Data of Peak Area and Peak Height Measurements.

Conc.

(mg/mL) Methanol:THF^a Precision (%)^b Accuracy (%)^c Area Height Area Height

76.1 6:0 0.69 0.35 101.18 102.30

5:1 0.20 2.00 101.67 81.71

4:2 0.44 0.99 100.59 64.77

3:3 0.37 0.22 103.13 53.91

133.1 6:0 0.67 0.65 101.30 101.16

5:1 0.81 1.18 101.16 82.47

4:2 0.79 0.32 100.94 65.28

3:3 0.49 0.39 101.32 54.31

199.7 6:0 0.36 1.16 100.36 101.34

5:1 0.37 1.59 100.64 80.36

4:2 0.45 2.12 102.74 65.05

3:3 0.37 0.21 102.68 53.69

aTHF stands for tetrahydrofuran. Also, the methanol-water mixture was used as a mobile phase. ^b,cPrecision = relative standard deviation (the number of determinations = 5); Accuracy = 100×(measured value/

nominal value).

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peak area remained the same despite tetrahydrofuran-triggered changes in the peak shape of progesterone. This was a reason why reasonable accuracy and precision data were obtained with peak area measurements at various methanol:tetrahydrofuran ratios tested in this study.

To confirm the effect of tetrahydrofuran on progesterone peak, an injection volume of a progesterone standard solution (199.7^µg/mL) consisting of methanol and tetrahydrofuran (4:2) was changed from 10 to 50^µL. Fig. 5 shows the HPLC chromatograms of progesterone obtained after injection of different volumes of a sample solution. As an injection volume increased, not only peak fronting but also peak broadening occurred extensively. When 10 and 15^µL of the sample solution was injected onto our analytical column, their asymmetry

factors were close to 1 (Fig. 6). At injection volumes equal to or greater than 20^µL, however, asymmetry factors started to fall below 0.9. These results are consistent with those shown in Fig. 2 and 3. Our data also agree well with the general sup- position that not more than 20^µL of a sample solution is injected into a 150×4.6 mm column.

The strengths of sample solutions containing larger proportions of tetrahydrofuran are quite different from those of 2 mobile phases used in this study, which may be held account- able for the broadening and fronting of progesterone peak. It has been previously suggested that an injection solvent stron- ger than a mobile phase interferes with the adsorption of an analyte at the column head (Keunchkarian et al., 2006). In par- ticular, such a problem worsens at the expense of a larger injection volume. Another major effect might arise from a dif-

Figure 4. Dependence of the assay accuracy upon the composition of a sample solution and peak analysis. Progesterone standard solutions with known concentrations were analyzed by (A) peak area and (B) peak height methods. It was the ammonium acetate-acetonitrile mixture that was used as a mobile phase.

Figure 5. HPLC chromatograms of progesterone obtained after injection of different volumes of a 199.7µg/mL progesterone solution having a methanol:tetrahydrofuran ratio of 4:2. Its injection volume was changed from (a) 10 to (b) 15, (c) 20, (d) 25, (e) 30, (f) 40 and (g) 50µL. The methanol-water mixture was used as a mobile phase.

Figure 6. Relationship between the asymmetry factor and the injection volume of a 199.7µg/mL progesterone solution consisting of methanol and tetrahydrofuran at a volume ratio of 4:2.

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ference in viscosity between an injection sample solution and a mobile phase, as suggested elsewhere (Cherrak et al., 1997).

If summarized, tetrahydrofuran might make the sample solution be distinguished from the mobile phases in relation to viscosity and strength. As a rule of thumb, the matrix of a sample solution is recommended to be the same as the matrix of a standard solution and/or a mobile phase. Our study highlights that in case when their matrices are different from one another, such differences should be carefully evaluated in order to

assure the accuracy and precision of an assay.

Based on the discussions of the HPLC chromatographic characteristics described above, an analytical methodology for the determination of progesterone content of PLGA microspheres was optimized as follows: 4 mL of tetrahydrofuran was used to dissolve progesterone-loaded microspheres, and 1 part of the solution was diluted with 5 parts of methanol to precipitate out the PLGA polymer. After removal of the poly- meric precipitates, 20 ^µL of the resultant solution having a methanol to tetrahydrofuran ratio of 5:1 was injected onto the analytical column. Fig. 7 and 8 show microencapsulation efficiencies of progesterone into PLGA microspheres prepared using ethyl fluoroacetate and ethyl chloroacetate, respectively.

The peak area determination substantiated that progesterone was almost completely encapsulated into PLGA microspheres.

Irrespective of microsphere formulations, microencapsulation efficiencies ranged from 93.63±2.97% to 101.63±0.59%.

However, the peak height measurement led to the underestimation of the microencapsulation efficiency of progesterone:

the values fell between 79.99±2.66% and 87.36±0.69%. Such false information deduced from the peak height measurement should be detrimental to developing a right dosage regimen.

Conclusion

The magnitudes of fronting, asymmetry and height reduction of progesterone peak are influenced by the proportion of tetrahydrofuran in a sample solution and the kind of a mobile phase used for elution. Furthermore, changes in such peak properties of progesterone become exacerbated at the expense of large injection volumes. The practical effect of tetrahydrofuran on the quality of peak size and peak shape is man- ifested with quantifying progesterone by area and height measurements. Therefore, the composition of a sample solution, the type of a mobile phase, and peak analysis methods should be carefully considered to achieve an accurate and reliable measurement of drug microencapsulation efficiency.

Acknowledgments

The authors thank Ms. J. Kim for preparing PLGA microspheres. This study was in part supported by a grant from the Korean Health Technology R&D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea (A092018).

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Figure 7. The percentage microencapsulation efficiency of progesterone determined by peak area and peak height measurements. Eth- yl fluoroacetate was used as a dispersed solvent to make PLGA microspheres. The amounts of progesterone used for microencapsulation varied from 60 to 100, 160, 200, and 250 mg.

Figure 8. The percentage microencapsulation efficiency of progesterone determined by peak area and peak height measurements. Eth- yl chloroacetate was used as a dispersed solvent to make PLGA microspheres. The amounts of progesterone used for microencapsulation varied from 60 to 100, 160, 200, and 250 mg.

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