Poly (D,L-lactide-co-glycolide) (PLGA) (50:50) with terminal carboxylate groups (PLGA, inherent viscosity 0.18 dl/g in hexafluoroisopropanol) was purchased from Absorbable Polymers International (Pelham, AL, USA). Poly (D,L-lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) di-block co-polymer (50:50 PLGA attached to mPEG 5000, 15% wt) was obtained from Evonik Degussa Corp (Birmingham, AL, USA). Docetaxel and paclitaxel were purchased from LC laboratories (Woburn, MA, USA). All reagents were analytical grade or above and used as received, unless otherwise stated.

Nanoparticles were prepared from PLGA and PLGA-PEG polymers using a modified emulsification solvent evaporation technique established in our lab. Briefly, the polymer was dissolved in ethyl acetate (10% w/v) and added into a 2.2% (w/v) PVA solution. The mixture was then vigorously shaken and subjected to high energy ultrasonication. The resulting nanosuspension was then subjected to stir for 2 hours to let the organic solvent evaporate. The nanoparticles were ultimately obtained after consecutive ultracentrifugation/washing steps. The obtained nanoparticles were finally resuspended in 1% sucrose aqueous solution and freeze-dried. The freeze- dried nanoparticles were stored at −20˚C for further use. Docetaxel-loaded PLGA or PLGA-PEG nanoparticles were prepared using different concentrations of drug through the same preparation approach. The various con- centrations of 0.25, 0.5, 1, and 1.5 mg/ml docetaxel solution were prepared in ethylacetate along with the poly- mer.

A stock solution of 100 µg/ml docetaxel was prepared by dissolving proper amount of drug in methanol and stored in −20˚C for further use. Working solution concentrations of 3.9, 7.8, 15.6, 31.2, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000, and 16,000 ng/ml were prepared by further dilution (serial) of the stock solution with methanol. A stock solution of paclitaxel in methanol as internal standard was also prepared at 100 µg/ml.

To prepare polymer-free standards (docetaxel in methanol), 10 µl of internal standard along with 1 ml of working solution (3.9 - 1000 ng/ml) were transferred into 2 ml tubes and vortexed. Polymer-free controls were prepared as such to obtain 100, 200, 400, and 800 ng/ml docetaxel concentrations. To prepare standard samples of nanoparticle formulations, 10 µl of internal standard along with 1 ml of working solution (125 - 8000 ng/ml and 125 - 16,000 ng/ml for PLGA-PEG and PLGA nanoparticles respectively) were transferred into 2 ml tubes and vortexed. The solvent was then evaporated and 5 mg plain (drug-free) nanoparticles were added to the tube. Concentration set of 1250, 2500, 5000, and 10,000 ng/ml was used to prepare quality control samples of PLGA nanoparticles. This is while 625, 1250, 2500, and 5000 ng/ml concentration set was used to prepare quality con- trol samples of PLGA-PEG nanoparticles. 10 µl of internal standard was added to control samples of PLGA or PLGA-PEG nanoparticles and vortexed. The solvent was evaporated and 5 mg plain nanoparticle was added to the tube. To prepare test samples, 10 µl of paclitaxel solution was added to 2 ml tubes along with 5 mg of drug loaded PLGA or PLGE-PEG nanoparticles. Standard, control, and test samples were extracted twice with ace- tone.

Docetaxel was extracted from PLGA or PLGA-PEG nanoparticles as follows. 1 ml acetone was added to 5 mg drug-loaded nanoparticles to dissolve both polymer and drug. The mixture was vortexed for 30 seconds and subjected to bath sonication for 30 minutes. Then it was centrifuged for 20 minutes at 20,000 g. The supernatant was separated and preserved. The precipitate was then dissolved in acetone and the same procedure was repeated. Obtained supernatants from the first and second centrifugation step were mixed and evaporated. 1 ml methanol was added to the residue, vortexed for 30 seconds and centrifuged for 20 minutes at 20,000 g. Docetaxel was then quantified in supernatant using mass spectrometry method.

Yield of the preparation method, drug loading, and encapsulation efficiency of nanoparticles were calculated as follows:

Yield (%) = (weight of obtained particles/initial weight of polymer, drug and other ingredients) × 100

Drug loading (%) = (weight of drug in particles/weight of particles) × 100

Encapsulation efficiency (%) = (weight of drug in particles/initial weight of drug added) × 100

Docetaxel and paclitaxel were solubilized (10 μg/mL) in methanol containing 0.1% (v/v) formic acid. Solutions were directly infused into the ionization source using a model 11 plus syringe pump (Harvard Apparatus, MA, USA) at a flow rate of 10 μL/min into a Hybrid Triple Quadrupole/Linear Ion trap mass spectrometer (AB Sciex 4000 QTRAP MS/MS system, Framingham, MA, US). The Turbo Ion-spray source was set to 5500 volts. Curtain gas, nebulizer, and heater gas pressure were 30, 40, and 40 psi respectively. The de-clustering potentials (DP) of docetaxel and paclitaxel were 46 and 55 respectively. Collision energys for docetaxel and paclitaxel were set to be 21 and 93 respectively. Collision cell exit potential was adjusted to 18 for both materials. Other instrument parameters include: interface heater = ON (150˚C), collisionally activated dissociation (CAD) = 5, and exit potential = 10. Ultimately, precursor ion scans (MS/MS) were performed using positive electrospray ionization (+ESI) with appropriate set mass. According to the obtained pattern of docetaxel and paclitaxel fragmentation, suitable fragments were chosen and used in MRM transitions. Injection of samples (10 µl) into the mass spectrometer was done using an Agilent Quaternary pump (1200 series) and Agilent G1329A (1200 series) auto sampler (Santa Clara, CA, USA) through a pre-column guard (Eclipse XDB-Rapid resolution, C18, 2.1 × 30 mm, 3.5 µ, Agilent, Santa Clara, CA, USA) with a mobile phase flow rate of 200 µl/min (methanol, 0.1% formic acid, isocractic elution). Quantitation procedure was performed on corresponding docetaxel and internal standard MRM Graphs using Analyst software version 1.6.

Positive ESI tandem mass spectra of docetaxel (809.4 m/z) and paclitaxel (855.2 m/z) are shown in Figure 1 and Figure 2 respectively. Large abundance of the parent ion was observed in the MS/MS spectra of both materials as protonated entities ([M + H]⁺). The proposed fragmentation pattern of each compound is also provided in upper right hand section of the figures. As exhibited in Figure 1, six product ions of docetaxel demonstrated high intensities. They include product ions with m/z of 226, 91, 105, 182, 528, and 119. Most abundant product ion was used in subsequent MRM transitions. Among those, product ion with m/z of 226 demonstrated the highest intensity. In case of paclitaxel (Figure 2), six product ions with m/z of 105, 106, 122, 286, 287, and 570 produced high intensities. Product ion with m/z of 105 provided the highest intensity. Depending on the experimental conditions, docetaxel and paclitaxel tend to generate different adducts when ionized through ESI source. Some studies have reported the alkali-adducts (mainly sodium adduct [M + Na]⁺) to be more intense (up to 5-fold) than protonated form ([M + H]⁺) [26] [37] . Although, there are reports that have exploited the protonated adduct

MRM transitions [38] [39] with good sensitivity. Taking into consideration the conditions used in this analysis method (e.g., isocractic elution), it was concluded that the protonated adduct could be the prominent ion. Consequently, in our study analysis was performed at the chosen MRM transitions in the presence of 0.1% v/v formic acid in methanol to obtain protonated adduct form the analyte and internal standard. Figure 3 demonstratestypical MRM Graphs of docetaxel and paclitaxel in various matrices.

For docetaxel solution in methanol, LOD and LOQ of the method were calculated to be 1.95 ng/ml and 3.9 ng/ml respectively. This is while docetaxel in PLGA and PLGA-PEG nanoparticles demonstrated LOD and LOQ values equal to 62.5 ng /ml and 125 ng /ml, respectively. The applied method covered a linearity range of 3.9 - 1000 ng/ml, 125 - 16,000 ng/ml, and 125 - 8000 ng/ml of docetaxel concentration respectively in methanol, PLGA nanoparticles and PLGA-PEG nanoparticles with a 1/x weighting factor. The method was sensitive enough to help evaluate the amount of drug in the abovementioned matrices. In all cases, obtained daily standard curves demonstrated regression correlation coefficients (R²) higher than 0.996. Analysis of docetaxel in methanol at LLOQ concentration (n = 6) demonstrated an accuracy and coefficient of variation (CV%) of less than 14.8% and 15.3% respectively. The evaluated within- and between-run variations of the docetaxel quantitation method in methanol (Table 1) demonstrated a CV%s less than 12.3% between concentration sets (i.e., good precision). Analysis of docetaxel in PLGA nanoparticels at LLOQ concentration (n = 6) exhibited an accuracy and CV% of less than 10.7% and 11.6% respectively. Within- and between-run variation studies of the docetaxel quantitation method in PLGA nanoparticles (Table 2) demonstrated CV%s less than 10.5% between various concentrations (i.e., good precision). CV%s for within- and between-run variation studies of docetaxel quantitation method in PLGA-PEG nanoparticles was calculated to be less than 10.94% (Table 3). Analysis of docetaxel in PLGA-PEG nanoparticels at LLOQ concentration (n = 6) demonstrated an accuracy and CV% of less than 13.42% and 13.07% respectively. In addition, back calculation of docetaxel quality control concentrations (accuracy) in methanol, PLGA nanoparticles, and PLGA-PEG nanoparticles exhibited values with acceptable deviation from actual concentrations (i.e., less than 15% deviation).These results suggested that the present method could accurately and reproducibly (acceptable precision) measure docetaxel in methanol, PLGA nanoparticles, and PLGA-PEG nanoparticles.

Table 4 reports loading characteristics of different drug loaded nanoparticle formulations. It has been observed that increasing the drug concentration in organic solution increased the entrapment of docetaxel in PLGA nanoparticles. The reason could be attributed to interacting more number of drug molecules with PLGA when initial drug concentration is increased [15] [40] . However, increasing the concentration of drug from 0.25 mg/ml to 1.5 mg/ml, the percentage of encapsulation efficiency demonstrates a descending trend (i.e., 47.76% to 37.25%). Increment in initial preparation concentration of drug has resulted in increased drug loading in PLGA-PEG nanoparticle as well. Encapsulation efficiency of PLGA-PEG nanoparticles is also decreased from 96.1% to 59.3% when initial drug payload is increased from 0.25 mg/ml to 1.5 mg/ml. For both type of nanoparticles, it is proposed that initial high encapsulation efficiency (e.g., at 0.25 mg/ml) might be due to high interactions of drug with polymer relative to the initial drug amount until polymer matrix gets saturated with drug molecules as more drug is used in the preparation procedure [15] .

It should be emphasized that determining the amount of loaded drug inside polymeric matrix is a crucial step in characterization of nanoparticle. To date, docetaxel has been quantitatively assayed by means of different HPLC [15] - [17] [41] or UV [18] set-ups reporting different assay outcomes. for instance, Esmaeili et al. [12] reported their method LOD to be 50 ng/ml with a retention time around 12 min. Musumeci and colleagues [13] determined docetaxel loading in PLGA nanoparticles at 0.7 - 37.9 µg/ml standard concentration range with a method sensitivity of 10 ng/ml. in another study, elution period was 25 min and the retention time of drug was about 9 min [14] . In comparison, run and retention time of our assay were 2 and 0.6 minutes respectively which is far less than that obtained by other studies. This allows analysis of a high number of samples within an extremely short period of time with lowest level of expenses. Although there are several reports of docetaxel being quantitatively analysed in biological matrices (e.g., plasma) using rapid ESI-MS/MS method [21] - [30] , yet quantitative analysis of docetaxel in polymeric matrices of PLGA and PLGA-PEG nanoparticles through direct injection to mass spectrometer has not been reported elsewhere.