A simple spectrophotometric evaluation method for the hydrophobic anticancer drug paclitaxel

In this work, we demonstrate a simple spectrophotometry approach to more accurately quantify and measure paclitaxel (PTX) concentrations. PTX cannot be precisely quantiﬁed when mixed with an aqueous solvent, and carries the risk of undergoing crystal precipitation. It is likely that PTX undergoes numerous interactions with aqueous solvents and enters a supersaturated state due to its low solubility. Therefore, a quantitative method is required to measure PTX for quality control before clinical use. Although several high-performance liquid chromatography (HPLC) methods have been reported to date, not all medical facilities have a clinical laboratory with such HPLC devices and analysis techniques. Spectroscopy is a simple and convenient method; however, calibration standards are prepared with an organic solvent, such as methanol and acetonitrile, which, when mixed with PTX, can cause solvent eﬀects that lead to inaccurate results. We generated a calibration curve of PTX at various concentrations (40%, 50%, 60%, 70%, 80%, 90%, and 100%) of methanol and evaluated the relative error from HPLC results. The optimum methanol concentration for quantiﬁcation of PTX was 65.8%, which corresponded to the minimum relative error. The detection limit and quantiﬁcation limit were 0.030 μg/mL and 0.092 μg/mL, respectively. It was possible to predict the PTX concentration even when polyoxyethylene castor oil and anhydrous ethanol were added, as in the commercially available PTX formulation, by diluting 32-fold with saline after mixing. Our ﬁndings show that PTX can be more accurately quantiﬁed using a calibration curve when prepared in a methanol/water mixture without the need for special devices or techniques. Abstract 21 In this work, we demonstrate a simple spectrophotometry approach to more accurately quantify 22 and measure paclitaxel (PTX) concentrations. PTX cannot be precisely quantified when mixed 23 with an aqueous solvent, and carries the risk of undergoing crystal precipitation. It is likely that PTX undergoes numerous interactions with aqueous solvents and enters a supersaturated state 25 due to its low solubility. Therefore, a quantitative method is required to measure PTX for quality 26 control before clinical use. Although several high-performance liquid chromatography (HPLC) 27 methods have been reported to date, not all medical facilities have a clinical laboratory with such 28 HPLC devices and analysis techniques. Spectroscopy is a simple and convenient method; 29 however, calibration standards are prepared with an organic solvent, such as methanol and 30 acetonitrile, which, when mixed with PTX, can cause solvent effects that lead to inaccurate 31 results. We generated a calibration curve of PTX at various concentrations (40%, 50%, 60%, 32 70%, 80%, 90%, and 100%) of methanol and evaluated the relative error from HPLC results.


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105 Preparation of saturated PTX in saline 106 107 Approximately 2.5 mg of PTX was added to 10 mL of saline, vortexed for 30 s and mixed by 108 rotating for 24 h at 30 rpm at 4°C. The supernatant was collected after centrifuging at 9,000 × g 109 for 10 min at 4°C. 50 μg/mL) in methanol and the PTX-saturated saline solution, prepared above, were injected 116 (20 μL each) into an octadecylsilyl silica gel column (5 μm, φ4.6 mm × h250 mm, Osaka Soda, 117 Osaka, Japan) of an Elite LaChrom HPLC system (Hitachi High-Technologies, Tokyo, Japan) 118 with 50% acetonitrile aqueous solution as the mobile phase and a flow rate of 1.2 mL/min 119 . The eluent was monitored at 230 nm. The peak area of PTX in each 120 standard solution was measured and plotted against the PTX concentration to generate a 121 calibration curve. The concentration of PTX in each test specimen (the PTX-saturated saline 122 solution) was subsequently determined using the same conditions as those used to generate the 123 standard calibration curve. The concentration of PTX-saturated saline was also measured spectrophotometrically at a 129 wavelength of 230 nm using a calibration curve prepared based on the methanol dilution series 130 (40%, 50%, 60%, 70%, and 80%). This experiment was replicated three times at room 131 temperature. The quantitative concentrations were compared to those obtained using HPLC. 132 Accuracy (R) indicates the relative error, which was defined as the deviation from the HPLC 133 results, and was calculated as follows: where Cs is the quantitative concentration measured spectrophotometrically and Cr is the 138 concentration measured using HPLC. For quality control against the commercially available PTX formulation, the method was 144 examined in the presence of polyoxyethylene castor oil and anhydrous ethanol. First, 30 mg of 145 PTX was added to 2.5 mL of polyoxyethylene castor oil and 2.5 mL of anhydrous ethanol. The 146 mixture was diluted 100-fold with saline, as in clinical use, and then further diluted from 2-fold 147 to 1,024-fold (final: 200-fold to 102,400-fold dilution) with saline to prepare a 2-fold dilution 148 series. The absorbance of each diluted solution was measured spectrophotometrically at a 149 wavelength of 230 nm. The PTX concentration in each diluted solution was determined using the 150 HPLC method described above. Reference solutions were prepared in the same manner with the 151 exclusion of PTX, and the absorbance was measured spectrophotometrically at a wavelength of 152 230 nm. Injectable PTX formulation was quantitatively analyzed using the method established in this 158 study, the accuracy of which was verified. The PTX formulation was diluted 100-fold with 159 saline, as in clinical use, and then further diluted 32-fold (final: 3,200-fold dilution) with saline. 160 This further 32-fold dilution is unique to this study and showed reasonable values in 161 spectroscopy. The PTX concentration was measured spectrophotometrically in the same manner 162 as that described above and compared to the results obtained using HPLC. The experiment was 163 replicated five times and the unpaired t-test was performed using Excel 2010 (Microsoft, 164 Redmond, WA, USA). Differences between the spectrometry and HPLC values were considered 165 statistically significant when the p-value was less than 0.05. We found that parameters of the PTX calibration curve, including slope and intercept, varied 173 depending on the solvent used ( Fig. 2 and Table 1). The calibration curves showed high 174 absorbance values at low concentrations of less than 1 μg/mL when the methanol concentration 175 was 80% or higher. While the values obtained at 50% and 60% methanol were comparable, a 176 marked change in the calibration curve was observed at 70-80% methanol. The findings suggest 177 that quantification results for PTX, particularly at low concentrations, may vary substantially if 178 experiments are conducted in solvents that differ to those used for calibration. According to HPLC, the average concentration of triplicate tests in the PTX-saturated saline 184 solution was 0.731±0.0438 μg/mL. The concentration in the same PTX-saturated saline solution 185 was also measured spectrophotometrically using each calibration curve, as shown in Fig. 2. The 186 absorbance of the PTX-saturated saline solution at a wavelength of 230 nm was 0.058-0.074, 187 which could not be measured using calibration curves prepared based on dilution in 90% or 188 100% methanol. Table 2 shows the spectroscopic quantitative concentrations calculated using 189 each calibration curve, including the accuracy (R). An R value close to "0" indicates that the 190 results from the two methods are comparable.
191 Figure 3 shows the correlation between the methanol concentration and relative error from the 192 HPLC results. The x-intercept of the approximate curves indicates the concentration of methanol 193 at which PTX concentrations in saline were comparable to those obtained using HPLC. 194 However, because the x-intercept could not be determined in this study, the solution to the 195 approximate curve equation, 65.8%, corresponding to the minimum relative error (-0.0174; Fig.  196 3, arrow), was identified as the optimum methanol concentration for quantification of PTX. The 197 calibration curve prepared using 0.313, 0.625, 1.25, and 2.50 μg/mL PTX in 65.8% methanol as 198 the solvent can therefore be expressed using the regression curve (r 2 = 0.9998) with the slope 199 0.0486 and the intercept 0.0032 (Fig. S1). The detection limit and quantification limit of PTX 200 were 0.030 μg/mL and 0.092 μg/mL, respectively.  Figure 4A shows the 209 absorbance at each dilution with or without PTX and the difference in absorbance, which would 210 indicate the absorbance derived from PTX. Figure 4B shows the PTX concentration in each 211 dilution compared to that obtained using HPLC, which was calculated using the differential 212 absorbance obtained in Fig. 4A and a calibration curve prepared using 65.8% methanol, the 213 optimal concentration for quantification of PTX. There was higher correlation between the 214 results obtained using the calibration curve and HPLC at a 32-fold dilution (dilution rate: 0.0313; 215 Fig. 4B, arrow) or less of the PTX concentration. These findings indicate that evaluation of the 216 PTX concentration in a test specimen should be conducted for quality control by mixing at a 217 dilution rate of 0.0313 (32-fold dilution).

Chemistry Journals
Analytical, Inorganic, Organic, Physical, Materials Science 222 According to the simulation described above, the commercially available PTX formulation can 223 be quantitatively analyzed without the HPLC method. First, PTX after mixing (100-fold dilution 224 with saline) should be further diluted 32-fold with saline before spectrophotometrically 225 measuring the absorbance at a wavelength of 230 nm (As), which produced a reading of 226 0.372±0.0168 in this study. The absorbance of the reference solution without PTX at a dilution 227 rate of 0.0313 (32-fold dilution) should be unchanged between lots and can be measured in 228 advance (Ar), producing a reading of 0.307±0.00814 in this study. The difference between As 229 and Ar (As -Ar: 0.0654±0.0168) would provide the absorbance of PTX in the test specimen at a 230 dilution rate of 0.0313, and the PTX concentration (1.28±0.346 μg/mL) can subsequently be 231 determined using a calibration curve prepared based on dilution in 65.8% methanol. Comparison 232 with the results obtained using HPLC showed that there was no significant difference between 233 values (Fig. 5). Fig. 6 shows a schematic illustration of the methodology established in this 234 study.

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237 Discussion 238 239 PTX can be more accurately quantified using a calibration curve when prepared in a 240 methanol/water mixture without the need for special devices or techniques. Solvent interactions 241 have been extensively studied since the 1970s and are known to affect not only the solubility but 242 also the stability and reaction rate of a solute (Hynes, 1985;Reichardt, 1982). Therefore, it is 243 important to evaluate the interactions of hydrophobic drugs such as PTX in polar aqueous 244 solvents. In most cases, calibration standards for hydrophobic drugs are prepared in non-polar 245 solvents, aprotic polar solvents, and certain alcohols. However, these standards may not be 246 accurate when evaluated using a spectrophotometer due to changes in the absorbance spectra as a 247 result of fundamental solute/solvent interactions or other factors. For verification, methanol and 248 acetonitrile with higher permeability in the target wavelength region (230 nm in this study) may 249 be more suitable than DMSO and DMF. In particular, methanol is less expensive and more 250 friendly to the environment than acetonitrile. Figure 2 shows that a marked change in the 251 calibration curve was particularly observed at 70-80% methanol. Wakisaka and Ohki showed 252 that the hydrogen bonding network (cluster level) in alcohol/water mixtures changes depending 253 on the alcohol concentration, and causes various solvent effects (Wakisaka & Ohki, 2005). They 254 found marked cluster-level changes at alcohol concentrations of 5.00-52.3% and 79.3-100%. As 255 the alcohol concentration increased, clusters of water molecules formed in the former range, 256 while clusters of alcohol molecules formed in the 52.3-79.3% range, and then disappeared in the 257 latter range. Because a lipophilic solute is more stable when surrounded by clusters of alcohol 258 molecules, PTX is expected to be more stable at methanol concentrations between 52.3% and 259 79.3%. We therefore speculate that the absorbance value increased at higher methanol 260 concentrations due to instability, leading to marked changes in the calibration curve. Our 261 findings suggest that quantification results for PTX, particularly at low concentrations, may vary 262 substantially if experiments are conducted in solvents that differ to those used for calibration.

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As shown in Fig. 3, we evaluated the relative error from HPLC results, and 65.8% was 264 identified as the optimum methanol concentration for quantification of PTX. The calibration 265 curve prepared using 0.313, 0.625, 1.25, and 2.50 μg/mL PTX in 65.8% methanol as the solvent 266 can therefore be expressed using the regression curve with the slope 0.0486 and the intercept 267 0.0032.

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On the other hand, PTX formulations contain polyoxyethylene castor oil and anhydrous ethanol, 269 and the effects of these solvents should be considered when quantifying PTX. As shown in Fig. 4, 270 there was higher correlation between the results obtained using the calibration curve and HPLC 271 at a 32-fold dilution or less of the PTX concentration. These findings indicated that it was 272 possible to predict the PTX concentration even when polyoxyethylene castor oil and anhydrous 273 ethanol were added, as in the commercially available PTX formulation, by diluting 32-fold with 274 saline after mixing, and the accuracy of the methods established in this study (Fig. 6) was 275 verified using the commercially available PTX formulation (Fig. 5).

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Although the results may differ depending on the chain length of the polyoxyethylene castor oil, 277 the theory and process should be similar for measuring other PTX solutions. While the Beer-278 Lambert law supports use of the additivity of absorbance for each component in the mixture, in 279 practice, it is necessary to verify whether the drugs and other components follow the law of 280 additivity of absorbance, including the presence or absence of interactions, even if they are 281 commercially available in mixed form, such as PTX formulations. To overcome the need to 282 verify this in our present study, we established an effective method for quantifying PTX in the 283 supersaturated state in saline based on correlations with the HPLC results, and determined the 284 required conditions for measurement using a calibration curve. We evaluated a simple and rapid method for determining the concentration of PTX in aqueous 290 solvent using spectrophotometry. Use of a calibration curve prepared based on dilution in 65.8% 291 methanol was effective for analyzing the PTX concentration in saline while minimizing the 292 solvent effect. Even when polyoxyethylene castor oil and anhydrous ethanol were added, as in 293 the commercially available PTX formulation, it was possible to predict the PTX concentration by 294 diluting 32-fold after mixing. This approach may be useful for quality control of PTX before 295 clinical use.