Facile one-pot green solvent synergized fluorescence reaction for determination of doxorubicin in presence of paclitaxel; co-administered drug, application to stability study and analysis in bulk, vial and biological fluids

2.7.1 Analysis of DOX in human plasma

The authors got the permission for using plasma sample of human volunteers from Minia Hospital according to the guidelines of the Commission on the Ethics of Scientific Research, Faculty of Pharmacy, Minia University. Code number for the approval of the Project is14/2020. In all cases, informed written consent was obtained from all participants. Into series a heparinized tubes, 5.0 mL of human blood free from the drug were centrifuged for 30 min. at 4000 rpm. 2 mL aliquots of supernatant plasma were transferred into a series of centrifugation tubes. Aliquots of standard solution of DOX were added so that the final concentration is in the range of 0.05–4 µg ml⁻¹. The tubes were mixed well and then suitable amount of acetonitrile was added to precipitate the protein (Derayea et al., 2018). The tubes were swirled for 60 s, and then centrifuged at 4000 rpm for 10 min. One milliliter of the clear supernatant was then quantitatively transferred into 5-mL calibrated volumetric flasks, and the previously mentioned procedure under “General analytical procedure” was applied. A blank experiment was performed simultaneously. Samples of human plasma spiked with different amounts of DOX in the working concentration range, were treated similarly, and the found contents of each added concentration of the drug were determined using its corresponding regression equation.

2.7.2 Analysis of DOX in human urine

Fresh urine sample from healthy people was filtrated and the filtrate was treated in the same manner as plasma, then aliquots of treated filtrate was pipetted into a 5-mL calibrated volumetric flask, and general procedure was followed for determination of DOX.

2.9.1 Alkaline and acidic degradation

Aliquots of DOX stock solutions containing 100 μg DOX were transferred into a series of 10-mL calibrated volumetric flasks, followed by the addition of 2 mL of 0.1 M HCl or 0.1 M NaOH. The solutions were incubated at 80 °C in a thermostatically controlled water bath for different time intervals (15–135 min). At each time interval, the content of each flask was neutralized to pH 7.0 using 0.1 M NaOH or 0.1 M HCl, respectively, and the volume was completed to the mark with ethanol. The induced degradation in both alkaline or acidic medium was carried out in a dark place to exclude the possible interfering from the light degradation effect. Accurate portions of 0.5 mL of the final solutions were transferred into 5-mL calibrated volumetric flasks and procedure was completed as mentioned under ‘General analytical procedure’.

2.9.2 Oxidative degradation

The solution for oxidative degradation study was prepared by precisely transferring 5 mL of DOX stock solution (100 μg ml⁻¹) into 50 mL calibrated volumetric flask, and the volume was reached to mark with aqueous H₂O₂ (3.0% v/v) solution. The solution was incubated at 80 °C in a thermostatically controlled water bath. A suitable volume of this solution containing 10 μg ml⁻¹ of DOX was quantitatively added to a 5-ml volumetric flask at different particular time intervals every 15 min. for 135 min. The general analytical procedure was applied to assay the remaining intact undamaged DOX.

2.9.3 Photo-degradation

UV Degradation: At a distance of 15 cm., a 10 mL well fitted stoppered glass tube containing DOX working solution (10 μg ml⁻¹) was holed in a metal box and exposed to UV emitting Lamp (254 nm) for 10 hrs., blank samples were prepared similarly but protected from light by being wrapped in aluminum foil, and the procedure was completed as mentioned in ‘General analytical procedure’.

Degradations with direct sunlight: A well fitted stoppered glass tube (10 mL) containing DOX working solution (10 μgml⁻¹) was exposed to direct sunlight for 4 hrs. Blank samples were prepared in the same manner but protected from light by being wrapped with aluminum foil, and the procedure was completed as mentioned in ''General analytical procedure“ at each time interval of 1 h to assay the remaining intact drug in presence of degraded molecules.

Daylight degradation: A volume of DOX solution was exposed to daylight (not exposed to direct sunlight) for 10 hrs., blank was prepared in similar manner but protected from daylight by being rolled in aluminum foil, and the general procedure was carried out.

3.1.1 Effect of different surfactant media

In attempt to intensify the fluorescence characters of DOX solution, a numerous micellar media were tried including; anionic surfactant (SDS), cationic surfactant (HTAB), non-ionic surfactants, (PEG 6000, and Tween-80), anionic polysaccharide (CMC Na) and others macromolecules as β-cyclodextrine and PVP. It was found that the anionic polysaccharide, CMC Na, has no significant effect while slight increase in the fluorescence was observed in the case of PEG 6000, PVP and β-CD. However, tween-80 and SDS and HTAB system yielded approximately two folds increase on the native fluorescence intensity of the drug compared with water (Fig. 3.).

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Fig. 3

The effect of different media and solvent on the fluorescence intensity of DOX (1.0 µg mL⁻¹).

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3.1.2 Effect of diluting solvent

Many solvents were used for final dilution, including; methanol, ethanol, acetonitrile, distilled water, dimethylsulfoxide; (DMSO) and dimethylformamide (DMF). It was observable that there was a marked increase in the fluorescence intensity of DOX with all of the investigated organic solvents in comparison with the obtained effect from the previously studied micellar media (Fig. 3). From all of the tested solvents, ethanol achieved the highest intensity of the intrinsic fluorescence. The obtained fluorescence intensity was inversely propositional with the polarity of the solvent in the following order; ethanol (1347), methanol (1022), DMF (986), acetonitrile (994), DMS (1024) and water (397). The values of dielectric constants of tested solvents were: 24.5, 32.7, 36.7, 37.5, 46.7 and 80.1 for ethanol, methanol, DMF, acetonitrile, DMSO and water respectively. A good linear correlation coefficient (r = 0.9134) was obtained between the fluorescence intensity and the dielectric constants of the diluting solvent. Consequently, ethanol was the sensitizing solvent of choice in the present study for quantification of DOX, which gave a higher fluorescence enhancement even than those achieved with any other solvent or organized media.

3.1.3 Effect of pH and volume

The effect of pH on the fluorescence intensity was examined over a wide pH range of 1.0–10.0 using hydrochloric acid (pH 1.0–2.0) and Britton-Robinson buffer solutions (2.0–10.0). It was found that, fluorescence activity of the drug was highly pH dependent. The highest enhancement in the measured fluorescence intensity was obtained in the acidic media (pH 1.0–2.0). Increasing or decreasing the pH apart from this range produced a distinct decrease in the intensity of fluorescence as shown in Fig. 4a. The same result was concluded from varying the volume of 0.5 M HCl where the best results were attained by using volumes of 0.5 M hydrochloric acid between 0.5 and 1.5 mL as shown in Fig. 4b. Therefore the optimum pH was 1.5 which was adjusted by using 1.0 mL of 0.5 M hydrochloric acid solution.

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Fig. 4

Effect of pH (a) and volume of 0.5 HCl (b) on fluorescence intensity of 1.0 µg mL⁻¹ DOX.

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3.2.1 Range and linearity

By analyzing a series of different concentrations of the standard solution covering the linear range of DOX (3 replicates for each concentration); the linearity of the developed method was estimated. After plotting the intensity of fluorescence against the corresponding drug concentration; a linear relationship was obtained, over the range of 7 × 10⁻⁴–4.0 µg mL⁻¹. The determination coefficient (r²), and correlation coefficient (r) were 0.9998 and 0.9999, respectively, indicating the excellent linearity of the developed method. Table 1 summarized the calculated statistical parameters for determination of DOX by the proposed method. Calibration was also performed in biological samples. The calibration curves in plasma and urine samples were constructed using six different concentration levels. In this case, the lower limit of quantitation (LLOQ) was defined as the lowest concentration on the calibration curve that can be determined with acceptable accuracy (80–120%) and precision (within 20%). The upper limit of quantitation (ULOQ) was identical to the highest calibration curve point that could be determined with accepted sensibility. The linear range for tested biological fluids was ranged from 0.05 to 4.0 µg mL⁻¹. The resulted regression equation for plasma analysis was y = 1257.2x + 13.6 with the 0.999 and 0.998 for r and r² respectively. For urine analysis the equation was y = 1289.2x + 23.28 with the 0.998 and 0.997 for r and r² respectively.

3.2.2 Quantitation (LOQ) and detection (LOD) limits

Through applying the equations: LOQ = 10Sa/b and LOD = 3.3Sa/b; and according to the recommendation of ICH Q2 (R1) (Guideline, 2005), the LOD and LOQ were calculated. In these equation; b is the slope of calibration curve, and Sa is the standard deviation of the intercept. The values of LOD and LOQ were 0.2 and 0.6 µg mL⁻¹, respectively, which indicate the high sensitivity of the present method for DOX estimation.

3.2.3 Accuracy

For an accuracy checking, five standard drug solutions with different concentration levels (0.05, 0.5, 1.0, 2.0 and 3.0 μg mL⁻¹) were analyzed by the proposed method in three replicates. The percentage recovery and standard deviation were calculated as shown in Table 2. The values of found recoveries percent were close to 100% with very low percentage error (Er.% < ± 0.5) which impart the high accuracy of developed methods. This is an acceptable level for the routine analysis of the studied drug.

3.2.4 Precision

The precision of the present method was tested at two levels; intra-day and inter-day levels. Intra-day precision was examined by performing the general analytical procedure to determine three concentrations of standard DOX solution (0.05, 0.5 and 1.0 µg mL⁻¹) during the same day. The same three previous drug concentrations were also estimated in the inter-day level, but by carrying out the analytical procedure over a period of three successive days. The analysis was done in three replicates at each concentration for both precision levels. The results were displayed in the form of percentage recovery and relative standard deviation (RSD) as illustrated in Table 3. The found data reflected the high precision of the present method as imparted by the low values of RSD.

3.2.5 Robustness

Minor variations in the experimental conditions that may influence the analytical performance were intentionally done to check the robustness of the presented method. As the method was very simple, only two parameters needed to be examined; pH, volume of HCl solution. As shown in Table 4, the changes have no significant effect on either the percentage recovery or standard deviation and the obtained values were within the recommended concentration. This implies an evidence for the excellent degree of robustness of the established method, and its reliability.

3.2.6 Selectivity

The selectivity of the present procedure was investigated by determination of cited DOX drug in the presence of paclitaxel (PTX); which is an adjuvant drug used in the chemotherapy course as first line for treatment of many solid tumors. The method appeared very acceptable results in different mixtures without any notable influence of PTX in quantification of DOX. It is noteworthy that, the analysis was made in both bulk powder and pharmaceutical dosage form. As shown in Table 5, the calculated recovery were 97.4–100.57, while the values of RSD were 0.28–2.12; These results imply that there is no interference observed in the determination of DOX at the studied concentrations. Therefore, the present procedure could selectively and effectively determine DOX in the presence of PXT.

3.2.7 Application to pharmaceutical formulations

The commercially available dosage form of DOX; adricin® vial (as a representative example) was subjected for quantification of its drug content by the proposed method and the reported one (Sastry and Rao, 1996). The student's t- and F-tests were applied to statistically compare the results obtained from both methods (Table 6). The calculated values of both tests are less than the tabulated one which point out that the results of both the proposed and reported methods don't differ significantly. However, the proposed procedure has the merit over the reported one in being more sensitive, simple, less time consuming utilizing no harmful or staining reagents and having very small detection limits. The results confirm that the developed procedure is suitable for the cited drug quantification with high recoveries, and the absence of interference from considerably involved dosage form additives. Therefore, the method is a proper choice for the analysis dosage forms containing the cited drug and for the routine analysis in quality control and quality assurance (see Table 5).

3.2.8 Application to spiked human plasma and urine

Because of the sufficient sensitivity of the presented method, it could be used to determine DOX in biological fluids such as human plasma and urine. The result for the application of the proposed method, are shown in Table 7. The percentage recoveries for plasma and urine analysis were in the range of 97.52–99.48% with a relative standard deviation of 0.88–2.03. These results indicate the ability of the current method for the quantitative measurement of the cited drug in human plasma and urine without any significant interference from the matrix of these biological samples.

3.3.1 Alkali induced degradation

The study revealed that the drug showed a high liability for alkaline degradation, represented by the significant decrease in the fluorescence intensity when heated with 0.1 M NaOH solution at 80 °C for more than 2 h. in thermostatically controlled water bath. Under the previously mentioned conditions, about 87.8% of the drug was degraded after the first hour. Four degradation products were separated and identified by mass spectroscopy in a previously reported study (Štenglová Netíková et al., 2017); however, the main and more stable one is shown in Fig. 5. A proposal for degradation of DOX under the different conditions is shown in Fig. 5.

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Fig. 5

Suggested alkaline (Štenglová Netíková et al., 2017), acidic (Wassermann and Bundgaard, 1983) and oxidative degradation (Kaushik and Bansal, 2015) pathways for the DOX.

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3.3.2 Acid induced degradation

Studying the influence of induced acidic condition on the stability of DOX showed moderate liability when the drug was incubated in 0.1 M HCl for more than 2 h at 80 °C in thermostatically controlled water bath (about 19.9% degradation after 1 h.). DOX is consists of tetracyclic quinoid aglycone; linked to amino sugar together through a glycosidic bond. Like other glycosides, doxorubicin is susceptible to undergo hydrolytic degradation in acidic solution with formation of the aglycone and the amino sugar as shown in Fig. 5 (Wassermann and Bundgaard, 1983).

3.3.3 Induced oxidative degradation

DOX undergone slight breakdown under oxidative conditions as confirmed by the slight decrease in fluorescence intensity after heating with 3% v/v H₂O₂ solution at 80 °C for 2 h. Only about 9.7% degradation was recorded upon exposing DOX solution to the mentioned oxidative condition for 1 h. As shown in Fig. 5, the oxidation could yield four degradation products as previously confirmed in a previous study (Kaushik and Bansal, 2015). The degradation occur through peroxide formation of the aliphatic side chain followed by either the complete removal of this side chain or further hydroxylation of the benzene ring at ortho- or para- position with respect to the methoxy group (Kaushik and Bansal, 2015).

3.3.4 Photolytic degradation

There is a wide variation about the photo-degradation of DOX in many of the previously published studies. Some of these studies have shown that the drug is resistant to photolytic degradation (Kaushik and Bansal, 2015). While there are other studies that prove that the drug is light sensitive (Wood et al., 1990; Bandak et al., 1999; Le Bot et al., 1987).

According to the present study, DOX showed a considerable degree of instability in daylight. When an aqueous solution of DOX was exposed to daylight for 10 h. about 62.4% degradation was observed. However, a similar solution stored in the dark for the same period showed 44.1% degradation. In addition, when the ethanolic solution of the drug was exposed to daylight for the same time experienced, only 22.4% degradation of the drug was observed. Based on these results, one could conclude that the daylight degradation in an aqueous solution is a mixed pathway including the photo-degradation and the hydrolytic effect. The same behavior was also observed when the drug solution was exposed to direct sunlight. However, sunlight produced a severe degradation and the drug was completely broken within two hours. To exclude the effect of hydrolysis, sun light degradation was carried out in absolute ethanol where about 76.03% of the drug was degraded within 4 h. On the other hand, DOX drug solution showed good stability to UV radiation. The observed degradation with UV-irradiation for 10 h, was 37.7% which was very close to the degree of degradation (32.9%) that occurred upon storing the drug in the dark for the same period.

The current study gives an indication of the relative stability of DOX towards oxidizing agents and UV degradation. While it was found that the drug is highly sensitive to hydrolysis, especially in the alkaline medium and photolysis with direct sunlight. A summary for the degradation profile of DOX under different condition is presented in Table 8.

3.3.5 Kinetics study of the degradation

The acidic, alkaline and oxidative degradations were noticed to be time-related, Fig. 6. The apparent first-order degradation rate constants were calculated using the equation: log A_o/A = kt/2.303, where A_o and A are the initial and remaining concentrations of DOX, respectively k is the reaction rate constant and t is the time of degradation. The value of k would equal the slope/2.303 and the half lifetime t_1/2 = 0.693/k. The calculated rate constants (k) were 0.0038, 0.021 and 0.0019 K h⁻¹ and half times (t_1/2) were 3.05, 0.56 and 6.26 h for acidic, alkaline and oxidative degradations, respectively .

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Fig. 6

Influence of different incubation times with 0.1 M HCl, 0.1 M NaOH and 3.0% v/v H₂O₂ on the DOX degradation rate (1 μg ml ⁻¹).

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