RP-HPLC-UV Method for Estimation of Fluorouracil–Epirubicin–Cyclophosphamide and Their Metabolite Mixtures in Human Plasma (Matrix)

RP-HPLC-UV Method for Estimation of Fluorouracil–Epirubicin–Cyclophosphamide and Their... Abstract A combination of 5-fluorouracil (FU), epirubicin (EP) and cyclophosphamide (CP) is routinely employed in the treatment of breast cancer. The objective of this study was to develop a reverse phase high-performance liquid chromatography (HPLC)-UV method for simultaneous quantitative analysis of the triple-drug and their metabolites in plasma. RP-HPLC system with a C18 column and a diode array detector was employed. The plasma samples were precipitated with acetonitrile and the supernatant was dried under a flow of nitrogen gas. The mobile phase comprised of two combinations, water (pH 4.0) and methanol (98:2 v/v), and water (pH 4.0):methanol:acetonitrile (70:13:17 v/v/v). The retention times for the compounds were determined and the parameters of validation established in plasma indicated the robustness and reliability. The corresponding HPLC peaks were confirmed using electron spray ionization mass spectrometry. FU and metabolites had a recovery of >93%; EP, epirubicinol and CP were >78% from plasma. Stability at 28–30°C in water (pH 4.0) of FU, 5,6-dihydro-5-fluorouracil and EP were higher followed by CP, EPol, fluorodeoxyuridine and fluorouridine (FUR). Storage of the drug-spiked plasma at −80°C assessed for 72 h showed a small but significant (P < 0.05) change in the recovery of FUR and EP. The method was validated in patient's plasma samples (n = 6). Introduction In 1976, it was first reported that chemotherapy with the combination of cyclophosphamide, methotrexate and 5-fluorouracil (CMF) was effective in treating women with locally advanced breast cancer. In an attempt to improve on the results of the CMF, anthracycline containing drugs were studied. Doxorubicin, an anthracycline drug was used initially which was then replaced by epirubicin, wherein the latter was less cardiotoxic without any compromise on antitumor efficacy. Hence, an intensive regimen of 5-FU, epirubicin (EP) and cyclophosphamide (CP), FEC for the treatment of node-positive and locally advanced breast cancer was established (1). Treatment with FEC has been shown to be safe and active first-line treatment for metastatic breast cancer (2). 5-FU is a fluoropyrimidine, antimetabolite drug which acts by misincorporating fluoronucleotide into RNA and DNA and by inhibiting the enzyme thymidylate synthase (TS). It is used widely in the treatment of malignancies of breast, gastrointestinal tract, head and neck. The side effects of 5-FU include hematological, mucosal and gastrointestinal toxicity (3). The hydrogen atom at the C5 of uracil is replaced by the fluorine in 5-FU (Figure 1a). It enters the cell by the facilitative transport mechanism of uracil (4). There are two routes, competing with each other—the anabolic route, which gives rise to the active metabolites, and the catabolic route, which inactivates 5-FU and leads to its elimination from the system (4). In the anabolic route, there are mainly three active metabolites of 5-FU namely, fluorodeoxyuridine monophosphate (FdUMP), fluorodeoxyuridine triphosphate (FdUTP) and fluorouridine triphosphate (FUTP). The activation of 5-FU involves its conversion to fluorouridine monophosphate (FUMP) either directly or via fluorouridine (FUR) (5). In the catabolic route, up to 80% of the 5-FU administered is reduced to 5,6-dihydro-5-fluorouracil (DHFU) by the enzyme dihydropyrimidine dehydrogenase (DPD). This is the rate-limiting step of 5-FU catabolism in normal and tumor cells (Supplementary Figure S1). Though fluoro-β-alanine (FBAL) is the major catabolite of 5-FU, fluoride ion (F–), N-carboxy-α-fluoro-β-alanine (CFBAL), three conjugates of FBAL with bile acids (2-fluoro-3-hydroxypropanoic acid [FHPA] and fluoroacetate [FAC]) formed by transamination of FBAL have also been reported as the catabolites of 5-FU (4). Figure 1. View largeDownload slide Chemical structure of 5-FU (a), EP (b) and CP (c). Figure 1. View largeDownload slide Chemical structure of 5-FU (a), EP (b) and CP (c). EP is an anthracycline drug used in treating broad spectrum of solid neoplasm and hematologic malignancies, non-small- and small-cell lung cancer, non-Hodgkin’s lymphoma, ovarian and gastric cancer (6, 7). EP is also known as 4′-epidoxorubicin, as it is an epimer of doxorubicin. The difference in the orientation of the C4′ hydroxyl group of EP and doxorubicin plays a major role in their metabolism (8, 9). Myelosuppression and cardiotoxicity are the major dose-limiting toxicity of anthracyclines (8). The orientation of C4′ hydroxyl group on EP (Figure 1b) makes it prone to liver glucuronidation, therefore it is more rapidly metabolized than doxorubicin at equimolar doses, which makes it relatively less toxic (9). Therefore, cumulative doses of EP for safe administration is between 950 and 1,000 mg/m2 (2). Other side effects of EP include, nausea, vomiting, alopecia, diarrhea and mucositis (8). EP is extensively distributed into the tissues and predominantly eliminated by biliary excretion. It is metabolized in the liver to 13-dihydro derivative, epirubicinol (EPol) by aldoketoreductase. EP and EPol conjugates with glucuronic acid forming corresponding glucuronides by the action of glucuronosyltransferases. Further hydrolysis leads to the formation of four aglycones (6) (Supplementary Figure S2). Cyclophosphamide is a nitrogen mustard belonging to the oxazophorines group (10) (Figure 1c). It is a prodrug and has limited cytotoxic or alkylating activity upon administration. On activation by liver microsomal enzymes, its cytotoxic and alkylating activities are enhanced (11). It adds an alkyl group to the DNA guanine base at N7 of imidazole (10). It has significant immunosuppressive activity and therefore used in treating autoimmune disorders and graft rejection (11). CP is hydroxylated at C4 by cytochrome P450 to form 4-hydroxy cyclophosphamide, which remains in dynamic equilibrium with aldophosphamide formed by the opening of the ring. Phosphoramide mustard and acrolein are then formed by cleavage of aldophosphamide. Phosphoramide mustard is involved in the DNA alkylation (12) (Supplementary Figure S3). Reverse phase high-performance liquid chromatography (RP-HPLC), which comprises a non-polar stationary phase and a relatively polar mobile phase has been employed previously for separation of the above-mentioned drugs. The detection methods employed for the drugs were primarily based on properties such as UV absorption, fluorescence and mass spectrometry. Based on the previous studies, we have attempted to develop a single, simple and reliable UV-HPLC method for quantitative analysis of the triple-drug combination (FEC) and their metabolites in plasma samples. For this, we used parent compounds 5-FU, CP, EP and drug metabolites DHFU, FUR, FUDR and EPol. Details of a combined RP-HPLC-UV-based method for the detection and quantitation of the above-mentioned compounds in plasma are presented. Experimental Instrumentation and reagents The chromatography system comprised of DionexUltiMate 3000 HPLC system, consisting of an ACC-3000 autosampler, column compartment, degasser, a column oven, a VWD-3000 variable wavelength detector and a DionexUltiMate 3000 diode array detector was procured from Dionex Systems, USA. C18 column (4.6 mm I.D., 150 mm length, 5 μm particle size) used for separation was purchased from Waters (USA). The data were analyzed using Chromeleon Chromatography Management System (Dionex, USA). The solvents used, double distilled and filtered water, Methanol (Sigma) and Acetonitrile (Fluka) were of HPLC grade. FU, FUR, FUDR and CP were purchased from MP Biomedicals (USA). DHFU, EP and EPol from Santa Cruz Biotechnology (USA). The mass spectrometry analysis was performed using Electron Spray Ionization-Trap Mass Spectrometer (amaZon ETD Ion Trap, Bruker Daltonics, Germany) equipped with ESI source (Bruker Daltonics, Germany). The syringe pump system was controlled by HyStar software (version 3.4, Bruker Daltonics, Germany) and the ion trap was controlled by EsquireControl software (version 7.0, Bruker Daltonics, Germany). Methods Patient samples About 5 mL of peripheral blood was collected from patients on obtaining appropriate informed consents, after 3 h of FU and EP injections and immediately after CP injection at the first cycle of FEC protocol, in vacutainer tubes coated with potassium salt of ethylenediaminetetraacetic acid (EDTA). Totally, samples were taken from six patients, of which two were administered FEC-60 protocol (5-FU 600, EP 60 and CP 600 mg/m2) and four under FEC-90 protocol (5-FU 600, EP 90 and CP 600 mg/m2). Similarly, drug-free samples were collected from patients upon obtaining informed consents. The sample collection from the patients was approved by the Institutional Ethical Committee. The plasma was separated by centrifugation at 2,500 ×g for 20 min and stored as aliquots of 0.6 mL in 1.5 mL protein Lobind tubes (Eppendorf, Germany) at −80°C until analysis. Chromatographic conditions Two different mobile phases were employed, for the initial 30 min, water (pH 4.0) and methanol (98:2 v/v) and from 31 to 60 min, water (pH 4.0), methanol and acetonitrile (70:13:17 v/v/v) and then till 80 min the former mobile phase was used. The pH of the water was adjusted using trichloroacetic acid (TCA) to pH 4.0. The injection volume was 5 μL, the flow rate 0.4 mL/min and column oven temperature was set at 27°C. The wavelengths of detection used were 195 , 200, 254, 265 and 270 nm. Sample preparation About 0.6 mL of plasma was thawed and centrifuged at 3,000 ×g to remove suspended matter. From this aliquots of 0.1 mL were taken in different 1.5 mL centrifuge tubes. To this, an equal volume of acetonitrile was added, mixed using vortex mixture and centrifuged at 12,000× g for 15 min. The supernatant was filtered through 0.22-μ spin filter and dried under a stream of nitrogen gas in Rapid mini EC system (Crescent Scientific, India) at 40°C for 30 min and reconstituted in 0.1 mL of water (pH 4.0). Preparation of stock and standard solutions Stock solution of FU, DHFU and FUR (1 mg/mL); FUDR (4 mg/mL); CP, EP and EPol (10 mg/mL) were prepared in methanol. Standard solutions of FU (2–10 μg/mL); DHFU (10–50 μg/mL); FUR (5–25 μg/mL); FUDR, EP and EPol (10–50 μg/mL) and CP (100–500 μg/mL) were prepared from the stock solution by diluting with water (pH 4.0). Linearity and recovery A calibration curve was plotted over the concentrations—FU (2, 4, 6, 8 and 10 μg/mL); DHFU, FUDR, EP and EPol (10, 20, 30, 40 and 50 μg/mL); FUR (5, 10, 15, 20 and 25 μg/mL) and CP (100, 200, 300, 400 and 500 μg/mL), in water (pH 4.0). The plasma samples were spiked with the drugs over the concentrations—FU (0.1, 1, 2, 4, 6, 8 and 10 μg/mL); DHFU, FUDR and EP (0.1, 1, 10, 20, 30, 40 and 50 μg/mL); EPol (1, 5, 10, 20, 30, 40 and 50 μg/mL), FUR (5, 10, 15, 20, 25, 100 and 200 μg/mL); and CP (10, 50, 100, 200, 300, 400 and 500 μg/mL) to standardize the protocol and study the linearity. The calibration curve equation was y = ax + b where y is the peak area, x is the concentration of each compound, a is the slope and b is the y-intercept of the graph. Recovery percentage was estimated by comparing the concentration obtained from the compounds in water (pH 4.0) and that in plasma at five concentrations within the above-mentioned range. Estimation of LOD and LOQ The lower limit of detection (LOD) and quantification (LOQ) was measured by using previously described method (13). Concentration corresponding to the peak with a signal-to-noise ratio of 3:1 was taken as LOD and that with a signal-to-noise ratio of 10:1 as LOQ. Precision and accuracy Precision of the method was measured as relative standard deviation (RSD %) for each compound at three different concentrations (lowest, medium and highest concentrations used in linearity experiments) using the formula, RSD % = (σ/mean) × 100 where σ is the standard deviation. Accuracy was calculated as the percentage of the ratio between nominal concentration and estimated concentration. Stability Stability of the compounds was tested by analyzing them at various time points—freshly prepared, after 1.5, 3, 5 and 24 h, upon storage at 28–30°C. The area under curve (AUC) was plotted against time (h) to demonstrate the stability of the compounds in water (pH 4.0) over the period of analysis. Stability of the compounds in plasma Stability of the compounds in plasma was analyzed by storing the spiked plasma samples at −80°C and carrying out the estimation as explained above at various time points. The plasma samples were precipitated and analyzed freshly, 24 , 48 and 72 h after spiking with three concentrations, lower, medium and higher concentrations, each in triplicates. The concentrations used were—FU (2, 6 and 10 μg/mL); DHFU (10, 30 and 50 μg/mL); FUR, FUDR and EP (10, 30 and 50 μg/mL); EPol (10, 20 and 50 μg/mL) and CP (100, 300 and 500 μg/mL). The significance of the difference was evaluated using Student's t-test. Validation of the peaks by mass spectrometry The peaks obtained using UV-HPLC were validated using ESI-MS. The analysis was performed by collecting the eluate from the column corresponding to the retention times (RT) of each compound. The collected eluent was dried under a stream of nitrogen gas using Rapid mini EC system (Crescent Scientific, India) at 40°C for 30 min and reconstituted in 10 μL water (LC-MS CHORMASOV®, Sigma-Aldrich, USA). This solution was introduced into the ESI source using a Hamilton syringe fixed to a syringe pump at a flow rate of 220 μL/h and the parameters used for measurements were capillary voltage −4,500 V; End Plate offset −500 V; dry gas 4.0 L/min; dry temperature 220°C; Nebulizer 8.0 psi and the scan mode was set to UltraScan mode and measurements were performed both in the positive (DHFU, EP, EPol and CP) and negative (FU, FUR and FUDR) ion mode. Results Chromatographic run conditions Based on previously published literature (Supplementary Tables 2–4), RP-HPLC-UV was chosen for determination of FU, DHFU, FUR, FUDR, EP and EPol and CP. In the process of developing a compatible assay for the triple-drug FEC and their metabolites, the mobile phase was optimized using different mixtures of water and organic solvents (methanol and acetonitrile) along with different column temperatures. During our method optimization routines, we found that the mobile phases previously used for EP and CP did not allow for the adequate RT of FU and its metabolites and as a result there was no chromatographic separation, and the compounds co-eluted with solvent peak, the HPLC-UV run optimization details of the analysis are summarized in Table I. On the other hand, RP-chromatography of CP and EP when performed under the conditions suitable for FU and its metabolites, we observed that the CP and EP failed to elute adequately despite extending the runtime and therefore could not be assessed, the HPLC-UV run optimization details for the analysis of CP and EP are summarized in Table II. Owing to the difference in the binding capacities of FU and its metabolites from that of EP, EPol and CP to stationary phase, two different compositions of the mobile phase were needed to ensure adequate separation of the compounds. Since our instrument was equipped with four pumps, it was possible to combine the two mobile phase mixtures, into a combined run with an overall runtime of 80 min. The mobile phase comprised of two sequential mixtures of reagents, the first was optimal for the separation of FU and its metabolites; and the second mixture was optimal for the separation of EP, EPol and CP. The chromatographic run was started with a mobile phase initially comprising water (pH 4.0) and methanol mixture (98:2 v/v) for the 30 min, which was optimal for the separation of FU and its metabolites, from 31 to 60 min mobile phase comprised of water (pH 4.0), methanol and acetonitrile mixture (70:13:17 v/v/v) optimal for the separation of EP, EPol and CP. The run finally ended (61–80 min) with a mobile phase comprised of water (pH 4.0) and methanol mixture (98:2 v/v). A flow rate of 0.4 mL/min and a column temperature of 27°C was found optimal for peak resolution. Table I. HPLC analysis of 5-FU and its metabolites using different mobile phases, column parameters and flow rate Sl no. Mobile phase (water at pH 4.0: MeOH: ACN) Column Result 1 70:7:3 C18, 5 μ, 150 mm at 40°C 5-FU and its metabolites could not be separated 2 70:7:3 C18, 5 μ, 250 mm at 40°C 5-FU and its metabolites could not be separated 3 80:20 (without ACN) C18, 5 μ, 250 mm at RT and 40°C Merged peaks of 5-FU and metabolites, FUR and FUDR 4 80:20 (without ACN) C18, 5 μ, 150 mm at RT and 40°C 5-FU and its metabolites eluted as distinct peaks but peak shape was poor at RT 5 90:0 (without ACN) C18, 5 μ, 150 mm at RT, 40°C and 50°C At 50°C, 5-FU and DHFU did not separate out. FU and DHFU peaks appeared as merged peaks 6 95:5 (without ACN) C18, 5 μ, 150 mm at 50°C FU and DHFU formed a double peak 7 98:2 (without ACN) C18, 5 μ, 150 mm at 50°C FU and its metabolites separated, but the RT was not consistent among repetitive runs 8 98:2 (without ACN) C18, 5 μ, 150 mm at 37°C FU and its metabolites separated Sl no. Mobile phase (water at pH 4.0: MeOH: ACN) Column Result 1 70:7:3 C18, 5 μ, 150 mm at 40°C 5-FU and its metabolites could not be separated 2 70:7:3 C18, 5 μ, 250 mm at 40°C 5-FU and its metabolites could not be separated 3 80:20 (without ACN) C18, 5 μ, 250 mm at RT and 40°C Merged peaks of 5-FU and metabolites, FUR and FUDR 4 80:20 (without ACN) C18, 5 μ, 150 mm at RT and 40°C 5-FU and its metabolites eluted as distinct peaks but peak shape was poor at RT 5 90:0 (without ACN) C18, 5 μ, 150 mm at RT, 40°C and 50°C At 50°C, 5-FU and DHFU did not separate out. FU and DHFU peaks appeared as merged peaks 6 95:5 (without ACN) C18, 5 μ, 150 mm at 50°C FU and DHFU formed a double peak 7 98:2 (without ACN) C18, 5 μ, 150 mm at 50°C FU and its metabolites separated, but the RT was not consistent among repetitive runs 8 98:2 (without ACN) C18, 5 μ, 150 mm at 37°C FU and its metabolites separated Table I. HPLC analysis of 5-FU and its metabolites using different mobile phases, column parameters and flow rate Sl no. Mobile phase (water at pH 4.0: MeOH: ACN) Column Result 1 70:7:3 C18, 5 μ, 150 mm at 40°C 5-FU and its metabolites could not be separated 2 70:7:3 C18, 5 μ, 250 mm at 40°C 5-FU and its metabolites could not be separated 3 80:20 (without ACN) C18, 5 μ, 250 mm at RT and 40°C Merged peaks of 5-FU and metabolites, FUR and FUDR 4 80:20 (without ACN) C18, 5 μ, 150 mm at RT and 40°C 5-FU and its metabolites eluted as distinct peaks but peak shape was poor at RT 5 90:0 (without ACN) C18, 5 μ, 150 mm at RT, 40°C and 50°C At 50°C, 5-FU and DHFU did not separate out. FU and DHFU peaks appeared as merged peaks 6 95:5 (without ACN) C18, 5 μ, 150 mm at 50°C FU and DHFU formed a double peak 7 98:2 (without ACN) C18, 5 μ, 150 mm at 50°C FU and its metabolites separated, but the RT was not consistent among repetitive runs 8 98:2 (without ACN) C18, 5 μ, 150 mm at 37°C FU and its metabolites separated Sl no. Mobile phase (water at pH 4.0: MeOH: ACN) Column Result 1 70:7:3 C18, 5 μ, 150 mm at 40°C 5-FU and its metabolites could not be separated 2 70:7:3 C18, 5 μ, 250 mm at 40°C 5-FU and its metabolites could not be separated 3 80:20 (without ACN) C18, 5 μ, 250 mm at RT and 40°C Merged peaks of 5-FU and metabolites, FUR and FUDR 4 80:20 (without ACN) C18, 5 μ, 150 mm at RT and 40°C 5-FU and its metabolites eluted as distinct peaks but peak shape was poor at RT 5 90:0 (without ACN) C18, 5 μ, 150 mm at RT, 40°C and 50°C At 50°C, 5-FU and DHFU did not separate out. FU and DHFU peaks appeared as merged peaks 6 95:5 (without ACN) C18, 5 μ, 150 mm at 50°C FU and DHFU formed a double peak 7 98:2 (without ACN) C18, 5 μ, 150 mm at 50°C FU and its metabolites separated, but the RT was not consistent among repetitive runs 8 98:2 (without ACN) C18, 5 μ, 150 mm at 37°C FU and its metabolites separated Table II. HPLC analysis of EP, CP and EPol, using different mobile phases, column parameters and flow rate Sl. No. Mobile phase (water at pH 4.0: MeOH: ACN) Column Result 1 70:5:25 C18, 5 μ, 150 mm at RT CP separated out, but merged peaks of epirubicin and epirubicinol 2 70:10:20 C18, 5 μ, 150 mm at RT CP separated out, but the separation of epirubicin and epirubicinol was poor 3 70:13:17 C18, 5 μ, 150 mm at RT CP separated out, but the separation of epirubicin and epirubicinol was good but epirubicinol peak shape poor 4 70:13:17 C18, 5 μ, 150 mm at 40°C CP, epirubicin and epirubicinol sharp peak and good separation Sl. No. Mobile phase (water at pH 4.0: MeOH: ACN) Column Result 1 70:5:25 C18, 5 μ, 150 mm at RT CP separated out, but merged peaks of epirubicin and epirubicinol 2 70:10:20 C18, 5 μ, 150 mm at RT CP separated out, but the separation of epirubicin and epirubicinol was poor 3 70:13:17 C18, 5 μ, 150 mm at RT CP separated out, but the separation of epirubicin and epirubicinol was good but epirubicinol peak shape poor 4 70:13:17 C18, 5 μ, 150 mm at 40°C CP, epirubicin and epirubicinol sharp peak and good separation Table II. HPLC analysis of EP, CP and EPol, using different mobile phases, column parameters and flow rate Sl. No. Mobile phase (water at pH 4.0: MeOH: ACN) Column Result 1 70:5:25 C18, 5 μ, 150 mm at RT CP separated out, but merged peaks of epirubicin and epirubicinol 2 70:10:20 C18, 5 μ, 150 mm at RT CP separated out, but the separation of epirubicin and epirubicinol was poor 3 70:13:17 C18, 5 μ, 150 mm at RT CP separated out, but the separation of epirubicin and epirubicinol was good but epirubicinol peak shape poor 4 70:13:17 C18, 5 μ, 150 mm at 40°C CP, epirubicin and epirubicinol sharp peak and good separation Sl. No. Mobile phase (water at pH 4.0: MeOH: ACN) Column Result 1 70:5:25 C18, 5 μ, 150 mm at RT CP separated out, but merged peaks of epirubicin and epirubicinol 2 70:10:20 C18, 5 μ, 150 mm at RT CP separated out, but the separation of epirubicin and epirubicinol was poor 3 70:13:17 C18, 5 μ, 150 mm at RT CP separated out, but the separation of epirubicin and epirubicinol was good but epirubicinol peak shape poor 4 70:13:17 C18, 5 μ, 150 mm at 40°C CP, epirubicin and epirubicinol sharp peak and good separation Chromatographic method validation To begin with, we assessed the reliability of chromatographic method to detect and quantitate FU, DHFU, FUDR, FUR, EP, EPol and CP. The method was performed as described in the previous section. The peaks of the compounds were picked by comparing with the blank profile. RT (mean ± SD) were as follows: for DHFU (λmax 200 nm) tr = 7.74 ± 0.019 min, FU (λmax 265 nm) tr = 10.51 ± 0.033 min, FUR (λmax 270 nm) tr = 22.66 ± 0.106, FUDR (λmax 270 nm) tr = 32.35 ± 0.109 min and EPol (λmax 254 nm) tr = 38.96 ± 0.087, EP (λmax 254 nm) tr = 40.81 ± 0.142 min, CP (λmax 195 nm) tr = 60.31 ± 0.069 (Figure 2). Calibration curve for standard solutions was plotted over the ranges stated in the previous section (Supplementary Figure S4). The regression equations were y = 6.697x + 2.070 (r2 = 0.9909 ± 0.0054) for FU, y = 3.092x + 4.038 (r2 = 0.9923 ± 0.0034) for DHFU, y = 1.187x – 5.127 (r2 = 0.9913 ± 0.0027) for FUDR, y = 0.7032 × – 0.5587 (r2 = 0.9918 ± 0.0039) for FUR, y = 0.1055 × – 0.0784 (r2 = 0.9963 ± 0.0002) for EP, y = 0.1078x – 0.0089 (r2 = 0.9880 ± 0.0009) for EPol, y = 0.0690x – 1.008 (r2 = 0.9910 ± 0.0045) for CP, respectively. For all the compounds, the coefficients of determination (r2 values) prove that the method was linear in the specified range for all the compounds tested. The results obtained for the calibration curve and the r2 values for the individual compounds are summarized in Table III. Figure 2. View largeDownload slide Chromatograms of blank and standard, respectively, of DHFU 30 μg/mL (a and b at 200 nm), FU 6 μg/mL (c and d at 265 nm), FUR 15 μg/mL and FUDR 30 μg/mL (e and f at 270 nm), EPol and EP both 30 μg/mL (g and h at 254 nm), CP 300 μg/mL (i and j at 195 nm). Figure 2. View largeDownload slide Chromatograms of blank and standard, respectively, of DHFU 30 μg/mL (a and b at 200 nm), FU 6 μg/mL (c and d at 265 nm), FUR 15 μg/mL and FUDR 30 μg/mL (e and f at 270 nm), EPol and EP both 30 μg/mL (g and h at 254 nm), CP 300 μg/mL (i and j at 195 nm). Table III. The retention times (RT) and other parameters of validation (linearity, LOD, LOQ, precision and accuracy) of the drugs and their metabolites in water (pH 4.0) Sl no. Compound RT (mean ± SD) LOD (ng/ml) LOQ (ng/ml) r2 Value (mean ± SD) Precision (RSD %) Accuracy (%) 1 FU 10.51 ± 0.033 2 10 0.9909 ± 0.0054 1.60 100.07 2 FUR 22.66 ± 0.106 30 150 0.9918 ± 0.0039 1.45 98.91 3 FUDR 32.35 ± 0.109 20 100 0.9913 ± 0.0027 0.53 99.47 4 DHFU 7.74 ± 0.019 10 50 0.9923 ± 0.0034 1.52 100.10 5 EP 40.81 ± 0.142 30 150 0.9963 ± 0.0002 0.71 99.93 6 EPol 38.96 ± 0.087 10 50 0.9880 ± 0.0009 1.31 99.28 7 CP 60.31 ± 0.069 200 1000 0.9910 ± 0.0045 0.27 99.85 Sl no. Compound RT (mean ± SD) LOD (ng/ml) LOQ (ng/ml) r2 Value (mean ± SD) Precision (RSD %) Accuracy (%) 1 FU 10.51 ± 0.033 2 10 0.9909 ± 0.0054 1.60 100.07 2 FUR 22.66 ± 0.106 30 150 0.9918 ± 0.0039 1.45 98.91 3 FUDR 32.35 ± 0.109 20 100 0.9913 ± 0.0027 0.53 99.47 4 DHFU 7.74 ± 0.019 10 50 0.9923 ± 0.0034 1.52 100.10 5 EP 40.81 ± 0.142 30 150 0.9963 ± 0.0002 0.71 99.93 6 EPol 38.96 ± 0.087 10 50 0.9880 ± 0.0009 1.31 99.28 7 CP 60.31 ± 0.069 200 1000 0.9910 ± 0.0045 0.27 99.85 Table III. The retention times (RT) and other parameters of validation (linearity, LOD, LOQ, precision and accuracy) of the drugs and their metabolites in water (pH 4.0) Sl no. Compound RT (mean ± SD) LOD (ng/ml) LOQ (ng/ml) r2 Value (mean ± SD) Precision (RSD %) Accuracy (%) 1 FU 10.51 ± 0.033 2 10 0.9909 ± 0.0054 1.60 100.07 2 FUR 22.66 ± 0.106 30 150 0.9918 ± 0.0039 1.45 98.91 3 FUDR 32.35 ± 0.109 20 100 0.9913 ± 0.0027 0.53 99.47 4 DHFU 7.74 ± 0.019 10 50 0.9923 ± 0.0034 1.52 100.10 5 EP 40.81 ± 0.142 30 150 0.9963 ± 0.0002 0.71 99.93 6 EPol 38.96 ± 0.087 10 50 0.9880 ± 0.0009 1.31 99.28 7 CP 60.31 ± 0.069 200 1000 0.9910 ± 0.0045 0.27 99.85 Sl no. Compound RT (mean ± SD) LOD (ng/ml) LOQ (ng/ml) r2 Value (mean ± SD) Precision (RSD %) Accuracy (%) 1 FU 10.51 ± 0.033 2 10 0.9909 ± 0.0054 1.60 100.07 2 FUR 22.66 ± 0.106 30 150 0.9918 ± 0.0039 1.45 98.91 3 FUDR 32.35 ± 0.109 20 100 0.9913 ± 0.0027 0.53 99.47 4 DHFU 7.74 ± 0.019 10 50 0.9923 ± 0.0034 1.52 100.10 5 EP 40.81 ± 0.142 30 150 0.9963 ± 0.0002 0.71 99.93 6 EPol 38.96 ± 0.087 10 50 0.9880 ± 0.0009 1.31 99.28 7 CP 60.31 ± 0.069 200 1000 0.9910 ± 0.0045 0.27 99.85 Drug and drug metabolite analysis in plasma: method validation Various methods for the recovery of drugs from plasma had been screened using organic solvents and solid phase extraction. For instance, solid phase extraction from plasma or diluted plasma and elution in acetonitrile, or solid phase extraction from plasma and elution with water pH 4.0, methanol and acetonitrile—70:13:17 v/v/v, or solid phase extraction followed by elution in methanol, or extraction using TCA were assessed, the details of extraction and the drawbacks observed are tabulated in Supplementary Table 1. Finally, the current method, acetonitrile-based precipitation was found to be appropriate since it yielded less interfering peaks with increased recovery. The chromatograms of the compounds recovered from plasma as against their unspiked blanks are given in Figure 3. Figure 3. View largeDownload slide Chromatograms of unspiked and spiked plasma, respectively, of DHFU 30 μg/mL (a and b at 200 nm), FU 6 μg/mL (c and d at 265 nm), FUR 15 μg/mL and FUDR 30 μg/mL (e and f at 270 nm), EPol and EP both 30 μg/mL (g and h at 254 nm), CP 300 μg/mL (i and j at 195 nm). Figure 3. View largeDownload slide Chromatograms of unspiked and spiked plasma, respectively, of DHFU 30 μg/mL (a and b at 200 nm), FU 6 μg/mL (c and d at 265 nm), FUR 15 μg/mL and FUDR 30 μg/mL (e and f at 270 nm), EPol and EP both 30 μg/mL (g and h at 254 nm), CP 300 μg/mL (i and j at 195 nm). Linearity and recovery Standard curves for all the seven compounds were produced with known concentrations spiked in plasma FU (range 0.1–10 μg/mL); DHFU, FUDR and EP (range 0.1–50 μg/mL); EPol (range 1–50 μg/mL), FUR (range 5–200 μg/mL); and CP (range 10–500 μg/mL). Seven points were plotted with AUC values of these drugs against their respective concentrations and linear regression analysis performed on the resultant curves (Supplementary Figure S5). The regression equations were y = 7.0950 × –0.3059 (r2 = 0.9993 ± 0.0062) for FU, y = 1.5460 × –0.5348 (r2 = 0.9997 ± 0.0073) for DHFU, y = 2.0350 × + 1.7980 (r2 = 0.9970 ± 0.003) for FUDR, y = 0.9183 × + 2.1740 (r2 = 0.9998 ± 0.0034) for FUR, y = 0.0670 ×–0.1514 (r2 = 0.9820 ± 0.0004) for EP, y = 0.2251 × –0.2488 (r2 = 0.9985 ± 0.0006) for EPol and y = 0.0547 × + 0.4741 (r2 = 0.9977 ± 0.0029) for CP, respectively. For all the compounds, the coefficients of determination (r2 values) prove that the method was linear in the specified range for all the compounds tested. The results obtained for the calibration curve and the r2 values for the individual compounds are summarized in Table IV. The percentage recovery was calculated by comparing the drug concentration from spiked plasma samples and that from standard solutions, as determined from peak areas, FU and metabolites had a recovery of >93%, whereas that of EP, EPol and CP were >78%. The percentage recoveries for each compound from plasma are as given in Table IV. Table IV. The retention times (RT), linearity (r2 values) and percentage recovery of the drugs and their metabolites in matrix Sl no. Compound RT (mean ± SD) r2 Value (mean ± SD) % Recovery Precision (RSD %) Accuracy (%) 1 FU 10.51 ± 0.033 0.9993 ± 0.0062 99.79 1.61 98.9 2 FUR 22.66 ± 0.106 0.9998 ± 0.0034 93.56 1.48 99.06 3 FUDR 32.35 ± 0.109 0.9970 ± 0.003 98.93 1.79 98.26 4 DHFU 7.74 ± 0.019 0.9997 ± 0.0073 98.58 1.64 101.72 5 EP 40.81 ± 0.142 0.9820 ± 0.0004 78.9 1.18 99.77 6 EPol 38.96 ± 0.087 0.9985 ± 0.0006 78.35 0.54 99.75 7 CP 60.31 ± 0.069 0.9977 ± 0.0029 79.34 0.5 99.62 Sl no. Compound RT (mean ± SD) r2 Value (mean ± SD) % Recovery Precision (RSD %) Accuracy (%) 1 FU 10.51 ± 0.033 0.9993 ± 0.0062 99.79 1.61 98.9 2 FUR 22.66 ± 0.106 0.9998 ± 0.0034 93.56 1.48 99.06 3 FUDR 32.35 ± 0.109 0.9970 ± 0.003 98.93 1.79 98.26 4 DHFU 7.74 ± 0.019 0.9997 ± 0.0073 98.58 1.64 101.72 5 EP 40.81 ± 0.142 0.9820 ± 0.0004 78.9 1.18 99.77 6 EPol 38.96 ± 0.087 0.9985 ± 0.0006 78.35 0.54 99.75 7 CP 60.31 ± 0.069 0.9977 ± 0.0029 79.34 0.5 99.62 Table IV. The retention times (RT), linearity (r2 values) and percentage recovery of the drugs and their metabolites in matrix Sl no. Compound RT (mean ± SD) r2 Value (mean ± SD) % Recovery Precision (RSD %) Accuracy (%) 1 FU 10.51 ± 0.033 0.9993 ± 0.0062 99.79 1.61 98.9 2 FUR 22.66 ± 0.106 0.9998 ± 0.0034 93.56 1.48 99.06 3 FUDR 32.35 ± 0.109 0.9970 ± 0.003 98.93 1.79 98.26 4 DHFU 7.74 ± 0.019 0.9997 ± 0.0073 98.58 1.64 101.72 5 EP 40.81 ± 0.142 0.9820 ± 0.0004 78.9 1.18 99.77 6 EPol 38.96 ± 0.087 0.9985 ± 0.0006 78.35 0.54 99.75 7 CP 60.31 ± 0.069 0.9977 ± 0.0029 79.34 0.5 99.62 Sl no. Compound RT (mean ± SD) r2 Value (mean ± SD) % Recovery Precision (RSD %) Accuracy (%) 1 FU 10.51 ± 0.033 0.9993 ± 0.0062 99.79 1.61 98.9 2 FUR 22.66 ± 0.106 0.9998 ± 0.0034 93.56 1.48 99.06 3 FUDR 32.35 ± 0.109 0.9970 ± 0.003 98.93 1.79 98.26 4 DHFU 7.74 ± 0.019 0.9997 ± 0.0073 98.58 1.64 101.72 5 EP 40.81 ± 0.142 0.9820 ± 0.0004 78.9 1.18 99.77 6 EPol 38.96 ± 0.087 0.9985 ± 0.0006 78.35 0.54 99.75 7 CP 60.31 ± 0.069 0.9977 ± 0.0029 79.34 0.5 99.62 LOD and LOQ The LOD and LOQ of the compounds were studied in water at pH 4.0. The LOD and LOQ concentrations of the compounds are given in Table III. Our method was able to detect all the compounds in the nanogram/milliliter range except in CP where LOD was 0.2 μg/mL and LOQ was 1 μg/mL. Precision and accuracy The precision and accuracy were calculated as mentioned in the previous section for the compounds in both water (pH 4.0) and plasma. Precision (%RSD) was <2% for all compounds, both in water (pH 4.0) and plasma. Similarly, accuracy was >98% for all compounds (Tables III and IV). Stability The stability of each of these compounds in water at pH 4.0 was studied by analyzing at various time points over a period of 24 h. FU, DHFU and EP were the most stable among the compounds studied at room temperature, with a difference in AUC measured after 24 h, not more than 0.5 mAU*min. CP, EPol and FUDR were initially stable but the AUC values decreased thereafter. For instance, AUC of CP decreased from 23.62 mAU*min at 5 h to 6.10 mAU*min at 24 h, for EPol tailing of the peak was observed with decrease in AUC from 3.41 mAU*min to 1.85 mAU*min and for FUDR, the initial peak with AUC 37.68 mAU*min was not observed after 24 h. FUR was found to be stable for 3 h, with a sharp dip in AUC assessed at the end of 5 h (9.51–1.39 mAU*min) (Figure 4a and b). In total, the analysis indicated that stability of FU, DHFU and EP was higher followed by CP, EPol, FUDR and FUR. Figure 4. View largeDownload slide (a) Stability of FU, DHFU, FUR and FUDR in water (pH 4.0). Plotted time in hours (h) on the x-axis and area under curve (AUC) in mAU*mins on the x-axis and (b) stability of EP, EPol and CP in water (pH 4.0). Plotted time in hours (h) on the x-axis and area under curve (AUC) in mAU*min on the y-axis. Figure 4. View largeDownload slide (a) Stability of FU, DHFU, FUR and FUDR in water (pH 4.0). Plotted time in hours (h) on the x-axis and area under curve (AUC) in mAU*mins on the x-axis and (b) stability of EP, EPol and CP in water (pH 4.0). Plotted time in hours (h) on the x-axis and area under curve (AUC) in mAU*min on the y-axis. Validation of the peaks by mass spectrometry The UV absorption peaks corresponding to each of the compounds were confirmed using mass spectrometry. The fractions collected upon HPLC separation was analyzed in MS as described above. The m/z values thus obtained was compared with that of the pure compounds. FU, FUR and FUDR were measured in the negative mode as [M-H]− with m/z ratios, 128.90, 260.75 and 244.90, respectively. DHFU, EP, EPol and CP were measured in the positive mode as [M + H]+ with m/z ratios 132.86, 544.02 and 546.10, respectively (Figure 5). The mass tolerance for the measurement was set at ±0.5 Da. The m/z values estimated for the HPLC peak fraction coincided with the observed masses of the standard compounds confirming the identity of compounds eluting in the corresponding peaks (Table V). Figure 5. View largeDownload slide Validation of UV-HPLC peaks of FEC and their metabolites, namely, DHFU (a), FU (b), FUR (c), FUDR (d), EP (e), EPol (f) and CP (g). FU, FUR and FUDR were measured in the negative mode and DHFU, EP, EPol and CP in the positive mode (Cut-off ±0.5 Da). Figure 5. View largeDownload slide Validation of UV-HPLC peaks of FEC and their metabolites, namely, DHFU (a), FU (b), FUR (c), FUDR (d), EP (e), EPol (f) and CP (g). FU, FUR and FUDR were measured in the negative mode and DHFU, EP, EPol and CP in the positive mode (Cut-off ±0.5 Da). Table V. Validation of UV-HPLC peaks of FEC and their metabolites by MS analysis Sl no. Compound RT min (mean ± SD) ESI Mode Expected mass (m/z) Observed mass (peak fraction) (m/z) (±0.5 Da) 1 FU 10.51 ± 0.033 − 129.01 [M−H]− 128.90 [M−H]− 2 DHFU 7.74 ± 0.019 + 133.04 [M+H]+ 132.86 [M+H]+ 3 FUR 32.35 ± 0.109 − 261.06 [M−H]− 260.75 [M−H]− 4 FUDR 22.66 ± 0.106 − 245.06 [M−H]− 244.90 [M−H]− 5 EP 40.81 ± 0.142 + 544.18 [M+H]+ 544.02 [M+H]+ 6 EPol 38.96 ± 0.087 + 546.19 [M+H]+ 546.10 [M+H]+ 7 CP 60.31 ± 0.069 + 261.03 [M+H]+ 260.71 [M+H]+ Sl no. Compound RT min (mean ± SD) ESI Mode Expected mass (m/z) Observed mass (peak fraction) (m/z) (±0.5 Da) 1 FU 10.51 ± 0.033 − 129.01 [M−H]− 128.90 [M−H]− 2 DHFU 7.74 ± 0.019 + 133.04 [M+H]+ 132.86 [M+H]+ 3 FUR 32.35 ± 0.109 − 261.06 [M−H]− 260.75 [M−H]− 4 FUDR 22.66 ± 0.106 − 245.06 [M−H]− 244.90 [M−H]− 5 EP 40.81 ± 0.142 + 544.18 [M+H]+ 544.02 [M+H]+ 6 EPol 38.96 ± 0.087 + 546.19 [M+H]+ 546.10 [M+H]+ 7 CP 60.31 ± 0.069 + 261.03 [M+H]+ 260.71 [M+H]+ Table V. Validation of UV-HPLC peaks of FEC and their metabolites by MS analysis Sl no. Compound RT min (mean ± SD) ESI Mode Expected mass (m/z) Observed mass (peak fraction) (m/z) (±0.5 Da) 1 FU 10.51 ± 0.033 − 129.01 [M−H]− 128.90 [M−H]− 2 DHFU 7.74 ± 0.019 + 133.04 [M+H]+ 132.86 [M+H]+ 3 FUR 32.35 ± 0.109 − 261.06 [M−H]− 260.75 [M−H]− 4 FUDR 22.66 ± 0.106 − 245.06 [M−H]− 244.90 [M−H]− 5 EP 40.81 ± 0.142 + 544.18 [M+H]+ 544.02 [M+H]+ 6 EPol 38.96 ± 0.087 + 546.19 [M+H]+ 546.10 [M+H]+ 7 CP 60.31 ± 0.069 + 261.03 [M+H]+ 260.71 [M+H]+ Sl no. Compound RT min (mean ± SD) ESI Mode Expected mass (m/z) Observed mass (peak fraction) (m/z) (±0.5 Da) 1 FU 10.51 ± 0.033 − 129.01 [M−H]− 128.90 [M−H]− 2 DHFU 7.74 ± 0.019 + 133.04 [M+H]+ 132.86 [M+H]+ 3 FUR 32.35 ± 0.109 − 261.06 [M−H]− 260.75 [M−H]− 4 FUDR 22.66 ± 0.106 − 245.06 [M−H]− 244.90 [M−H]− 5 EP 40.81 ± 0.142 + 544.18 [M+H]+ 544.02 [M+H]+ 6 EPol 38.96 ± 0.087 + 546.19 [M+H]+ 546.10 [M+H]+ 7 CP 60.31 ± 0.069 + 261.03 [M+H]+ 260.71 [M+H]+ Stability of the compounds in plasma stored at −80°C In order to assess the effects of sample storage on recovery of the parent drugs and metabolites from plasma, the spiked plasma samples were incubated for various time points over a period of 72 h, and analyzed in triplicates as described in the previous section. Three concentrations of the respective analyte ranging from low, medium and high levels were tested. There were no statistically significant differences in the concentration of the compounds (FU, DHFU, FUDR, EPol and CP) analyzed upon storage at −80°C for the given time periods. However, in the case of FUR and EP, values indicated a slight but statistically significant (P-value <0.05) decrease in the recovery at the higher concentration level at 72 h (Figure 6). Figure 6. View largeDownload slide Recovery of FU, DHFU, FUR, FUDR, EP, EPol and CP from matrix on storage at −80oC after 24 , 48 and 72 h. The samples were spiked with three concentrations of each drug (a–g) and concentration (μg/mL) was plotted against time (h). The values of FUR and EP showed a significant difference at higher concentration after 72 h (*P < 0.05). Figure 6. View largeDownload slide Recovery of FU, DHFU, FUR, FUDR, EP, EPol and CP from matrix on storage at −80oC after 24 , 48 and 72 h. The samples were spiked with three concentrations of each drug (a–g) and concentration (μg/mL) was plotted against time (h). The values of FUR and EP showed a significant difference at higher concentration after 72 h (*P < 0.05). Patient samples The analysis was performed in patient samples to assess the clinical applicability of our method. The samples from patients were collected after 3 h of FU and EP injections and immediately after CP injection at the first cycle of the protocol. The samples were analyzed immediately following isolation to avoid degradation as indicated from the stability analysis. Our method was able to detect the drugs and their metabolites in plasma of patients. FU was detected in all the samples (range 0.2–0.9 μg/mL), CP (range 66.1–159.7 μg/mL) and DHFU (range 0.30–1.2 μg/mL) were detected in all but one samples, FUDR (range 1.3–3.0 μg/mL) was seen in three and EP (0.6 and 0.2 μg/mL) in two samples. EPol and FUR were seen only in one of the samples tested (1.9 and 146.3 μg/mL, respectively) (Table VI). Table VI. The estimation of compounds (FU, EP—their metabolites and CP) in the plasma of patients undergoing chemotherapy with FEC regimen. Patient (↓) Dosage FU (μg/ml) DHFU (μg/ml) FUR (μg/ml) FUDR (μg/ml) EP (μg/ml) EPol (μg/ml) CP (μg/ml) 1 FEC-90 0.630 0.711 145.6 3.048 12.772 9.204 154.949 2 FEC-60 0.583 1.424 ND ND ND ND 132.729 3 FEC-90 0.276 1.543 ND 1.302 2.891 ND 115.755 4 FEC-60 0.336 ND ND ND ND ND 76.693 5 FEC-90 0.252 0.938 ND 1.650 ND ND ND 6 FEC-90 0.268 0.658 ND ND ND ND 95.614 Patient (↓) Dosage FU (μg/ml) DHFU (μg/ml) FUR (μg/ml) FUDR (μg/ml) EP (μg/ml) EPol (μg/ml) CP (μg/ml) 1 FEC-90 0.630 0.711 145.6 3.048 12.772 9.204 154.949 2 FEC-60 0.583 1.424 ND ND ND ND 132.729 3 FEC-90 0.276 1.543 ND 1.302 2.891 ND 115.755 4 FEC-60 0.336 ND ND ND ND ND 76.693 5 FEC-90 0.252 0.938 ND 1.650 ND ND ND 6 FEC-90 0.268 0.658 ND ND ND ND 95.614 ND—not detected. Table VI. The estimation of compounds (FU, EP—their metabolites and CP) in the plasma of patients undergoing chemotherapy with FEC regimen. Patient (↓) Dosage FU (μg/ml) DHFU (μg/ml) FUR (μg/ml) FUDR (μg/ml) EP (μg/ml) EPol (μg/ml) CP (μg/ml) 1 FEC-90 0.630 0.711 145.6 3.048 12.772 9.204 154.949 2 FEC-60 0.583 1.424 ND ND ND ND 132.729 3 FEC-90 0.276 1.543 ND 1.302 2.891 ND 115.755 4 FEC-60 0.336 ND ND ND ND ND 76.693 5 FEC-90 0.252 0.938 ND 1.650 ND ND ND 6 FEC-90 0.268 0.658 ND ND ND ND 95.614 Patient (↓) Dosage FU (μg/ml) DHFU (μg/ml) FUR (μg/ml) FUDR (μg/ml) EP (μg/ml) EPol (μg/ml) CP (μg/ml) 1 FEC-90 0.630 0.711 145.6 3.048 12.772 9.204 154.949 2 FEC-60 0.583 1.424 ND ND ND ND 132.729 3 FEC-90 0.276 1.543 ND 1.302 2.891 ND 115.755 4 FEC-60 0.336 ND ND ND ND ND 76.693 5 FEC-90 0.252 0.938 ND 1.650 ND ND ND 6 FEC-90 0.268 0.658 ND ND ND ND 95.614 ND—not detected. Discussion A study of the literature on the various methodologies developed revealed that there are HPLC-UV-based methods for the estimation of above-mentioned drugs and their metabolites either individually or as a combination in mixtures. Despite the fact that methods are available that combine FU and CP using HPLC-UV, the estimation of EP in most cases has been through a fluorescence detector (8, 14–17), and we were unable to identify a method that is suitable for the quantification of the triple-drug combination (FEC) and their metabolites simultaneously in plasma based on HPLC-UV (Supplementary Tables 2–4). In the case of FU the drug was estimated using a combination of gas chromatography–mass Spectrometry (18). In addition, metabolites of FU dihydrofluorouracil (DHFU), α-fluoro-β-ureidopropionic acid and α-fluoro-β-alanine (FBAL) were also estimated using gas chromatography–mass spectrometry (19). Studies had revealed the advantages of liquid chromatography based estimation of FU over gas chromatography, using 50 mM phosphate buffer at pH 3.0 as mobile phase and in addition also presented a tedious procedure for the extraction of drug from plasma (17, 20). A subsequent HPLC-UV method for the estimation of FU used a mobile phase comprising combination phosphate buffer and 8% methanol in phosphate buffer, pH 5.5 in a single run. Plasma extraction was by saturating with ammonium sulfate followed by n-propanol and ether-based extraction and the LOD of this method was 0.1 μg/mL (21). Similarly, studies have used a varying combination of mobile phases comprising phosphate buffer (3, 22), potassium phosphate–methanol (23), potassium dihydrogen phosphate and methanol (24), tetrabutylammonium hydrogen sulfate and potassium phosphate buffer (25). A summary of various mobile phases, detection wavelength and LOD and LOQ values reported for FU and metabolites are presented in Supplementary Table 2. In addition, fluorescence-based detection method was developed for FU and FUDR from the plasma of patients after derivatization using 3-bromomethyl-6, 7-dimethoxy-l-methyl-2(1H)-quinoxalinone (Br-DMEQ). The mobile phase used was simple, 35% methanol in water and the fluorescence was measured at excitation and emission wavelengths of 370 and 455 nm, respectively (26). Enzyme-based estimation have also been employed for instance FdUMP, an active antineoplastic metabolite of FU from tissues was measured based on the stoichiometric inhibition of Lactobacillus casei TS and was sensitive in detecting as low as 1 pmol of FdUMP (27). FU and the three metabolites DHFU, FUR and FUDR have been estimated using UV-HPLC method but their LOQ was much higher and in the μg/mL range. The LOQ was 0.5 μg/mL for FU, 1 μg/mL for DHFU, 3 μg/mL for FUR and FUDR. The relatively poor LOQ and recovery of this method could be attributed to the choice of detection wavelength. The study had chosen 210 nm for all the compounds, however, FU, FUR and FUDR have a maximum absorption in the range of 254–280 nm whereas DHFU has around 190 nm and hence, a compromise had to be made in case of having system that could detect only one wavelength (28) (Supplementary Table 2). Most of the studies on EP and EPol have been based on fluorescence detection at λex470–480 nm and λem550–560 nm (9, 29, 30) (Supplementary Table 3). Electron spray mass spectrometry has also been employed in the detection of EP, on elution with ammonium formate and acetonitrile at acidic pH of 3.0 (31). Very few studies are available for UV-HPLC method for estimation of EP, of which one of them used sodium formate, methanol and acetonitrile as mobile phase and detection wavelength was set at 280 nm (32). Another study used methanol and water as mobile phase and detection was at 254 nm. The LOD was 40 ng/mL, which is higher than that of our method (33). Cyclophosphamide and its metabolite have been estimated using methanol: 20 mM ammonium acetate containing 1% acetic acid (30:70 v/v) as mobile phase, and detection was mass spectrometry-based (34). Assay of cyclophosphamide in bulk drug used mobile phase of 10% acetonitrile as Solvent A and 70% acetonitrile as Solvent B and gradient was used for resolving the compound and the UV detection wavelength was set at 195 nm (10) (Supplementary Table 4). There are studies available in the literature that studied estimation of multiple drug combination by a UV-HPLC method (Supplementary Table 5). For instance, a study measured the three drugs 5-fluorouracil, adriamycin and cyclophosphamide with a mobile phase of 0.05 M disodium hydrogen phosphate and acetonitrile (65:35 v/v) at pH 3.7 and Diode Array detector set at 266 nm, 254 nm and 195 nm (17). FU and CP have also been measured in combination as part of the five antineoplastic agents using a mobile phase comprising water (pH 4.0), methanol, acetonitrile (70:13:17 v/v/v) and detection using DAD (16). Another plasma-based method for the estimation of FU and CP used the same mobile phase at the ratio 68:13:19 v/v/v (35). The LOD and LOQ of these methods ranged from 0.07–0.1 μg/mL and 0.098–1.0 μg/mL, respectively, for FU, whereas LOD and LOQ of our study was 2 and 10 ng/mL, respectively. The LOD and LOQ of CP were 0.1–0.3 and 0.15–0.5 μg/mL, respectively, lower than that in our study, 0.2 and 1 μg/mL, respectively. However, it must be noted that the multidrug estimation methodologies did not involve the drug EP in the mixture, hence a method was developed to estimate FEC triple-drug combination. In our estimations of drug levels in patient samples who were treated with FEC protocols, FEC-60 and FEC-90 where the dosage of FU and CP is the same with the difference being in the dose of EP, we observed that some drugs are observed at detectable levels while others are not. This variability in detection could be due to the influence of dose, genetic/clinico-pathological features, for instance, EP was observed in two patients from a total six and at a higher level in a FEC-90 relative to FEC-60. To understand the differences in observed values would require a larger series of samples to assess for the correlation of dose and genetic/clinico-pathological features with drug levels. Conclusion In summary, we have developed a UV-RP-HPLC method to estimate the parent compounds of the FEC regimen and some of their metabolites in plasma. In the future, this method could be employed in pharmacogenomic studies to study the metabolism of these drugs in patients treated with this regimen and possibly relate to their genotypes, which in turn can reveal clues to their differential toxicity/efficacy in the patient population. Supplementary data Supplementary material is available at Journal of Chromatographic Science online. Funding This study was funded by the Department of Science and Technology, Government of India. References 1 Levine , M.N. ; Randomized trial comparing cyclophosphamide, epirubicin, and fluorouracil with cyclophosphamide, methotrexate, and fluorouracil in premenopausal women with node-positive breast cancer: update of National Cancer Institute of Canada Clinical Trials Group Tr ; Journal of Clinical Oncology , ( 2005 ); 23 ( 22 ): 5166 – 5170 . 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Google Scholar CrossRef Search ADS © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Chromatographic Science Oxford University Press

RP-HPLC-UV Method for Estimation of Fluorouracil–Epirubicin–Cyclophosphamide and Their Metabolite Mixtures in Human Plasma (Matrix)

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
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0021-9665
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10.1093/chromsci/bmy020
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Abstract

Abstract A combination of 5-fluorouracil (FU), epirubicin (EP) and cyclophosphamide (CP) is routinely employed in the treatment of breast cancer. The objective of this study was to develop a reverse phase high-performance liquid chromatography (HPLC)-UV method for simultaneous quantitative analysis of the triple-drug and their metabolites in plasma. RP-HPLC system with a C18 column and a diode array detector was employed. The plasma samples were precipitated with acetonitrile and the supernatant was dried under a flow of nitrogen gas. The mobile phase comprised of two combinations, water (pH 4.0) and methanol (98:2 v/v), and water (pH 4.0):methanol:acetonitrile (70:13:17 v/v/v). The retention times for the compounds were determined and the parameters of validation established in plasma indicated the robustness and reliability. The corresponding HPLC peaks were confirmed using electron spray ionization mass spectrometry. FU and metabolites had a recovery of >93%; EP, epirubicinol and CP were >78% from plasma. Stability at 28–30°C in water (pH 4.0) of FU, 5,6-dihydro-5-fluorouracil and EP were higher followed by CP, EPol, fluorodeoxyuridine and fluorouridine (FUR). Storage of the drug-spiked plasma at −80°C assessed for 72 h showed a small but significant (P < 0.05) change in the recovery of FUR and EP. The method was validated in patient's plasma samples (n = 6). Introduction In 1976, it was first reported that chemotherapy with the combination of cyclophosphamide, methotrexate and 5-fluorouracil (CMF) was effective in treating women with locally advanced breast cancer. In an attempt to improve on the results of the CMF, anthracycline containing drugs were studied. Doxorubicin, an anthracycline drug was used initially which was then replaced by epirubicin, wherein the latter was less cardiotoxic without any compromise on antitumor efficacy. Hence, an intensive regimen of 5-FU, epirubicin (EP) and cyclophosphamide (CP), FEC for the treatment of node-positive and locally advanced breast cancer was established (1). Treatment with FEC has been shown to be safe and active first-line treatment for metastatic breast cancer (2). 5-FU is a fluoropyrimidine, antimetabolite drug which acts by misincorporating fluoronucleotide into RNA and DNA and by inhibiting the enzyme thymidylate synthase (TS). It is used widely in the treatment of malignancies of breast, gastrointestinal tract, head and neck. The side effects of 5-FU include hematological, mucosal and gastrointestinal toxicity (3). The hydrogen atom at the C5 of uracil is replaced by the fluorine in 5-FU (Figure 1a). It enters the cell by the facilitative transport mechanism of uracil (4). There are two routes, competing with each other—the anabolic route, which gives rise to the active metabolites, and the catabolic route, which inactivates 5-FU and leads to its elimination from the system (4). In the anabolic route, there are mainly three active metabolites of 5-FU namely, fluorodeoxyuridine monophosphate (FdUMP), fluorodeoxyuridine triphosphate (FdUTP) and fluorouridine triphosphate (FUTP). The activation of 5-FU involves its conversion to fluorouridine monophosphate (FUMP) either directly or via fluorouridine (FUR) (5). In the catabolic route, up to 80% of the 5-FU administered is reduced to 5,6-dihydro-5-fluorouracil (DHFU) by the enzyme dihydropyrimidine dehydrogenase (DPD). This is the rate-limiting step of 5-FU catabolism in normal and tumor cells (Supplementary Figure S1). Though fluoro-β-alanine (FBAL) is the major catabolite of 5-FU, fluoride ion (F–), N-carboxy-α-fluoro-β-alanine (CFBAL), three conjugates of FBAL with bile acids (2-fluoro-3-hydroxypropanoic acid [FHPA] and fluoroacetate [FAC]) formed by transamination of FBAL have also been reported as the catabolites of 5-FU (4). Figure 1. View largeDownload slide Chemical structure of 5-FU (a), EP (b) and CP (c). Figure 1. View largeDownload slide Chemical structure of 5-FU (a), EP (b) and CP (c). EP is an anthracycline drug used in treating broad spectrum of solid neoplasm and hematologic malignancies, non-small- and small-cell lung cancer, non-Hodgkin’s lymphoma, ovarian and gastric cancer (6, 7). EP is also known as 4′-epidoxorubicin, as it is an epimer of doxorubicin. The difference in the orientation of the C4′ hydroxyl group of EP and doxorubicin plays a major role in their metabolism (8, 9). Myelosuppression and cardiotoxicity are the major dose-limiting toxicity of anthracyclines (8). The orientation of C4′ hydroxyl group on EP (Figure 1b) makes it prone to liver glucuronidation, therefore it is more rapidly metabolized than doxorubicin at equimolar doses, which makes it relatively less toxic (9). Therefore, cumulative doses of EP for safe administration is between 950 and 1,000 mg/m2 (2). Other side effects of EP include, nausea, vomiting, alopecia, diarrhea and mucositis (8). EP is extensively distributed into the tissues and predominantly eliminated by biliary excretion. It is metabolized in the liver to 13-dihydro derivative, epirubicinol (EPol) by aldoketoreductase. EP and EPol conjugates with glucuronic acid forming corresponding glucuronides by the action of glucuronosyltransferases. Further hydrolysis leads to the formation of four aglycones (6) (Supplementary Figure S2). Cyclophosphamide is a nitrogen mustard belonging to the oxazophorines group (10) (Figure 1c). It is a prodrug and has limited cytotoxic or alkylating activity upon administration. On activation by liver microsomal enzymes, its cytotoxic and alkylating activities are enhanced (11). It adds an alkyl group to the DNA guanine base at N7 of imidazole (10). It has significant immunosuppressive activity and therefore used in treating autoimmune disorders and graft rejection (11). CP is hydroxylated at C4 by cytochrome P450 to form 4-hydroxy cyclophosphamide, which remains in dynamic equilibrium with aldophosphamide formed by the opening of the ring. Phosphoramide mustard and acrolein are then formed by cleavage of aldophosphamide. Phosphoramide mustard is involved in the DNA alkylation (12) (Supplementary Figure S3). Reverse phase high-performance liquid chromatography (RP-HPLC), which comprises a non-polar stationary phase and a relatively polar mobile phase has been employed previously for separation of the above-mentioned drugs. The detection methods employed for the drugs were primarily based on properties such as UV absorption, fluorescence and mass spectrometry. Based on the previous studies, we have attempted to develop a single, simple and reliable UV-HPLC method for quantitative analysis of the triple-drug combination (FEC) and their metabolites in plasma samples. For this, we used parent compounds 5-FU, CP, EP and drug metabolites DHFU, FUR, FUDR and EPol. Details of a combined RP-HPLC-UV-based method for the detection and quantitation of the above-mentioned compounds in plasma are presented. Experimental Instrumentation and reagents The chromatography system comprised of DionexUltiMate 3000 HPLC system, consisting of an ACC-3000 autosampler, column compartment, degasser, a column oven, a VWD-3000 variable wavelength detector and a DionexUltiMate 3000 diode array detector was procured from Dionex Systems, USA. C18 column (4.6 mm I.D., 150 mm length, 5 μm particle size) used for separation was purchased from Waters (USA). The data were analyzed using Chromeleon Chromatography Management System (Dionex, USA). The solvents used, double distilled and filtered water, Methanol (Sigma) and Acetonitrile (Fluka) were of HPLC grade. FU, FUR, FUDR and CP were purchased from MP Biomedicals (USA). DHFU, EP and EPol from Santa Cruz Biotechnology (USA). The mass spectrometry analysis was performed using Electron Spray Ionization-Trap Mass Spectrometer (amaZon ETD Ion Trap, Bruker Daltonics, Germany) equipped with ESI source (Bruker Daltonics, Germany). The syringe pump system was controlled by HyStar software (version 3.4, Bruker Daltonics, Germany) and the ion trap was controlled by EsquireControl software (version 7.0, Bruker Daltonics, Germany). Methods Patient samples About 5 mL of peripheral blood was collected from patients on obtaining appropriate informed consents, after 3 h of FU and EP injections and immediately after CP injection at the first cycle of FEC protocol, in vacutainer tubes coated with potassium salt of ethylenediaminetetraacetic acid (EDTA). Totally, samples were taken from six patients, of which two were administered FEC-60 protocol (5-FU 600, EP 60 and CP 600 mg/m2) and four under FEC-90 protocol (5-FU 600, EP 90 and CP 600 mg/m2). Similarly, drug-free samples were collected from patients upon obtaining informed consents. The sample collection from the patients was approved by the Institutional Ethical Committee. The plasma was separated by centrifugation at 2,500 ×g for 20 min and stored as aliquots of 0.6 mL in 1.5 mL protein Lobind tubes (Eppendorf, Germany) at −80°C until analysis. Chromatographic conditions Two different mobile phases were employed, for the initial 30 min, water (pH 4.0) and methanol (98:2 v/v) and from 31 to 60 min, water (pH 4.0), methanol and acetonitrile (70:13:17 v/v/v) and then till 80 min the former mobile phase was used. The pH of the water was adjusted using trichloroacetic acid (TCA) to pH 4.0. The injection volume was 5 μL, the flow rate 0.4 mL/min and column oven temperature was set at 27°C. The wavelengths of detection used were 195 , 200, 254, 265 and 270 nm. Sample preparation About 0.6 mL of plasma was thawed and centrifuged at 3,000 ×g to remove suspended matter. From this aliquots of 0.1 mL were taken in different 1.5 mL centrifuge tubes. To this, an equal volume of acetonitrile was added, mixed using vortex mixture and centrifuged at 12,000× g for 15 min. The supernatant was filtered through 0.22-μ spin filter and dried under a stream of nitrogen gas in Rapid mini EC system (Crescent Scientific, India) at 40°C for 30 min and reconstituted in 0.1 mL of water (pH 4.0). Preparation of stock and standard solutions Stock solution of FU, DHFU and FUR (1 mg/mL); FUDR (4 mg/mL); CP, EP and EPol (10 mg/mL) were prepared in methanol. Standard solutions of FU (2–10 μg/mL); DHFU (10–50 μg/mL); FUR (5–25 μg/mL); FUDR, EP and EPol (10–50 μg/mL) and CP (100–500 μg/mL) were prepared from the stock solution by diluting with water (pH 4.0). Linearity and recovery A calibration curve was plotted over the concentrations—FU (2, 4, 6, 8 and 10 μg/mL); DHFU, FUDR, EP and EPol (10, 20, 30, 40 and 50 μg/mL); FUR (5, 10, 15, 20 and 25 μg/mL) and CP (100, 200, 300, 400 and 500 μg/mL), in water (pH 4.0). The plasma samples were spiked with the drugs over the concentrations—FU (0.1, 1, 2, 4, 6, 8 and 10 μg/mL); DHFU, FUDR and EP (0.1, 1, 10, 20, 30, 40 and 50 μg/mL); EPol (1, 5, 10, 20, 30, 40 and 50 μg/mL), FUR (5, 10, 15, 20, 25, 100 and 200 μg/mL); and CP (10, 50, 100, 200, 300, 400 and 500 μg/mL) to standardize the protocol and study the linearity. The calibration curve equation was y = ax + b where y is the peak area, x is the concentration of each compound, a is the slope and b is the y-intercept of the graph. Recovery percentage was estimated by comparing the concentration obtained from the compounds in water (pH 4.0) and that in plasma at five concentrations within the above-mentioned range. Estimation of LOD and LOQ The lower limit of detection (LOD) and quantification (LOQ) was measured by using previously described method (13). Concentration corresponding to the peak with a signal-to-noise ratio of 3:1 was taken as LOD and that with a signal-to-noise ratio of 10:1 as LOQ. Precision and accuracy Precision of the method was measured as relative standard deviation (RSD %) for each compound at three different concentrations (lowest, medium and highest concentrations used in linearity experiments) using the formula, RSD % = (σ/mean) × 100 where σ is the standard deviation. Accuracy was calculated as the percentage of the ratio between nominal concentration and estimated concentration. Stability Stability of the compounds was tested by analyzing them at various time points—freshly prepared, after 1.5, 3, 5 and 24 h, upon storage at 28–30°C. The area under curve (AUC) was plotted against time (h) to demonstrate the stability of the compounds in water (pH 4.0) over the period of analysis. Stability of the compounds in plasma Stability of the compounds in plasma was analyzed by storing the spiked plasma samples at −80°C and carrying out the estimation as explained above at various time points. The plasma samples were precipitated and analyzed freshly, 24 , 48 and 72 h after spiking with three concentrations, lower, medium and higher concentrations, each in triplicates. The concentrations used were—FU (2, 6 and 10 μg/mL); DHFU (10, 30 and 50 μg/mL); FUR, FUDR and EP (10, 30 and 50 μg/mL); EPol (10, 20 and 50 μg/mL) and CP (100, 300 and 500 μg/mL). The significance of the difference was evaluated using Student's t-test. Validation of the peaks by mass spectrometry The peaks obtained using UV-HPLC were validated using ESI-MS. The analysis was performed by collecting the eluate from the column corresponding to the retention times (RT) of each compound. The collected eluent was dried under a stream of nitrogen gas using Rapid mini EC system (Crescent Scientific, India) at 40°C for 30 min and reconstituted in 10 μL water (LC-MS CHORMASOV®, Sigma-Aldrich, USA). This solution was introduced into the ESI source using a Hamilton syringe fixed to a syringe pump at a flow rate of 220 μL/h and the parameters used for measurements were capillary voltage −4,500 V; End Plate offset −500 V; dry gas 4.0 L/min; dry temperature 220°C; Nebulizer 8.0 psi and the scan mode was set to UltraScan mode and measurements were performed both in the positive (DHFU, EP, EPol and CP) and negative (FU, FUR and FUDR) ion mode. Results Chromatographic run conditions Based on previously published literature (Supplementary Tables 2–4), RP-HPLC-UV was chosen for determination of FU, DHFU, FUR, FUDR, EP and EPol and CP. In the process of developing a compatible assay for the triple-drug FEC and their metabolites, the mobile phase was optimized using different mixtures of water and organic solvents (methanol and acetonitrile) along with different column temperatures. During our method optimization routines, we found that the mobile phases previously used for EP and CP did not allow for the adequate RT of FU and its metabolites and as a result there was no chromatographic separation, and the compounds co-eluted with solvent peak, the HPLC-UV run optimization details of the analysis are summarized in Table I. On the other hand, RP-chromatography of CP and EP when performed under the conditions suitable for FU and its metabolites, we observed that the CP and EP failed to elute adequately despite extending the runtime and therefore could not be assessed, the HPLC-UV run optimization details for the analysis of CP and EP are summarized in Table II. Owing to the difference in the binding capacities of FU and its metabolites from that of EP, EPol and CP to stationary phase, two different compositions of the mobile phase were needed to ensure adequate separation of the compounds. Since our instrument was equipped with four pumps, it was possible to combine the two mobile phase mixtures, into a combined run with an overall runtime of 80 min. The mobile phase comprised of two sequential mixtures of reagents, the first was optimal for the separation of FU and its metabolites; and the second mixture was optimal for the separation of EP, EPol and CP. The chromatographic run was started with a mobile phase initially comprising water (pH 4.0) and methanol mixture (98:2 v/v) for the 30 min, which was optimal for the separation of FU and its metabolites, from 31 to 60 min mobile phase comprised of water (pH 4.0), methanol and acetonitrile mixture (70:13:17 v/v/v) optimal for the separation of EP, EPol and CP. The run finally ended (61–80 min) with a mobile phase comprised of water (pH 4.0) and methanol mixture (98:2 v/v). A flow rate of 0.4 mL/min and a column temperature of 27°C was found optimal for peak resolution. Table I. HPLC analysis of 5-FU and its metabolites using different mobile phases, column parameters and flow rate Sl no. Mobile phase (water at pH 4.0: MeOH: ACN) Column Result 1 70:7:3 C18, 5 μ, 150 mm at 40°C 5-FU and its metabolites could not be separated 2 70:7:3 C18, 5 μ, 250 mm at 40°C 5-FU and its metabolites could not be separated 3 80:20 (without ACN) C18, 5 μ, 250 mm at RT and 40°C Merged peaks of 5-FU and metabolites, FUR and FUDR 4 80:20 (without ACN) C18, 5 μ, 150 mm at RT and 40°C 5-FU and its metabolites eluted as distinct peaks but peak shape was poor at RT 5 90:0 (without ACN) C18, 5 μ, 150 mm at RT, 40°C and 50°C At 50°C, 5-FU and DHFU did not separate out. FU and DHFU peaks appeared as merged peaks 6 95:5 (without ACN) C18, 5 μ, 150 mm at 50°C FU and DHFU formed a double peak 7 98:2 (without ACN) C18, 5 μ, 150 mm at 50°C FU and its metabolites separated, but the RT was not consistent among repetitive runs 8 98:2 (without ACN) C18, 5 μ, 150 mm at 37°C FU and its metabolites separated Sl no. Mobile phase (water at pH 4.0: MeOH: ACN) Column Result 1 70:7:3 C18, 5 μ, 150 mm at 40°C 5-FU and its metabolites could not be separated 2 70:7:3 C18, 5 μ, 250 mm at 40°C 5-FU and its metabolites could not be separated 3 80:20 (without ACN) C18, 5 μ, 250 mm at RT and 40°C Merged peaks of 5-FU and metabolites, FUR and FUDR 4 80:20 (without ACN) C18, 5 μ, 150 mm at RT and 40°C 5-FU and its metabolites eluted as distinct peaks but peak shape was poor at RT 5 90:0 (without ACN) C18, 5 μ, 150 mm at RT, 40°C and 50°C At 50°C, 5-FU and DHFU did not separate out. FU and DHFU peaks appeared as merged peaks 6 95:5 (without ACN) C18, 5 μ, 150 mm at 50°C FU and DHFU formed a double peak 7 98:2 (without ACN) C18, 5 μ, 150 mm at 50°C FU and its metabolites separated, but the RT was not consistent among repetitive runs 8 98:2 (without ACN) C18, 5 μ, 150 mm at 37°C FU and its metabolites separated Table I. HPLC analysis of 5-FU and its metabolites using different mobile phases, column parameters and flow rate Sl no. Mobile phase (water at pH 4.0: MeOH: ACN) Column Result 1 70:7:3 C18, 5 μ, 150 mm at 40°C 5-FU and its metabolites could not be separated 2 70:7:3 C18, 5 μ, 250 mm at 40°C 5-FU and its metabolites could not be separated 3 80:20 (without ACN) C18, 5 μ, 250 mm at RT and 40°C Merged peaks of 5-FU and metabolites, FUR and FUDR 4 80:20 (without ACN) C18, 5 μ, 150 mm at RT and 40°C 5-FU and its metabolites eluted as distinct peaks but peak shape was poor at RT 5 90:0 (without ACN) C18, 5 μ, 150 mm at RT, 40°C and 50°C At 50°C, 5-FU and DHFU did not separate out. FU and DHFU peaks appeared as merged peaks 6 95:5 (without ACN) C18, 5 μ, 150 mm at 50°C FU and DHFU formed a double peak 7 98:2 (without ACN) C18, 5 μ, 150 mm at 50°C FU and its metabolites separated, but the RT was not consistent among repetitive runs 8 98:2 (without ACN) C18, 5 μ, 150 mm at 37°C FU and its metabolites separated Sl no. Mobile phase (water at pH 4.0: MeOH: ACN) Column Result 1 70:7:3 C18, 5 μ, 150 mm at 40°C 5-FU and its metabolites could not be separated 2 70:7:3 C18, 5 μ, 250 mm at 40°C 5-FU and its metabolites could not be separated 3 80:20 (without ACN) C18, 5 μ, 250 mm at RT and 40°C Merged peaks of 5-FU and metabolites, FUR and FUDR 4 80:20 (without ACN) C18, 5 μ, 150 mm at RT and 40°C 5-FU and its metabolites eluted as distinct peaks but peak shape was poor at RT 5 90:0 (without ACN) C18, 5 μ, 150 mm at RT, 40°C and 50°C At 50°C, 5-FU and DHFU did not separate out. FU and DHFU peaks appeared as merged peaks 6 95:5 (without ACN) C18, 5 μ, 150 mm at 50°C FU and DHFU formed a double peak 7 98:2 (without ACN) C18, 5 μ, 150 mm at 50°C FU and its metabolites separated, but the RT was not consistent among repetitive runs 8 98:2 (without ACN) C18, 5 μ, 150 mm at 37°C FU and its metabolites separated Table II. HPLC analysis of EP, CP and EPol, using different mobile phases, column parameters and flow rate Sl. No. Mobile phase (water at pH 4.0: MeOH: ACN) Column Result 1 70:5:25 C18, 5 μ, 150 mm at RT CP separated out, but merged peaks of epirubicin and epirubicinol 2 70:10:20 C18, 5 μ, 150 mm at RT CP separated out, but the separation of epirubicin and epirubicinol was poor 3 70:13:17 C18, 5 μ, 150 mm at RT CP separated out, but the separation of epirubicin and epirubicinol was good but epirubicinol peak shape poor 4 70:13:17 C18, 5 μ, 150 mm at 40°C CP, epirubicin and epirubicinol sharp peak and good separation Sl. No. Mobile phase (water at pH 4.0: MeOH: ACN) Column Result 1 70:5:25 C18, 5 μ, 150 mm at RT CP separated out, but merged peaks of epirubicin and epirubicinol 2 70:10:20 C18, 5 μ, 150 mm at RT CP separated out, but the separation of epirubicin and epirubicinol was poor 3 70:13:17 C18, 5 μ, 150 mm at RT CP separated out, but the separation of epirubicin and epirubicinol was good but epirubicinol peak shape poor 4 70:13:17 C18, 5 μ, 150 mm at 40°C CP, epirubicin and epirubicinol sharp peak and good separation Table II. HPLC analysis of EP, CP and EPol, using different mobile phases, column parameters and flow rate Sl. No. Mobile phase (water at pH 4.0: MeOH: ACN) Column Result 1 70:5:25 C18, 5 μ, 150 mm at RT CP separated out, but merged peaks of epirubicin and epirubicinol 2 70:10:20 C18, 5 μ, 150 mm at RT CP separated out, but the separation of epirubicin and epirubicinol was poor 3 70:13:17 C18, 5 μ, 150 mm at RT CP separated out, but the separation of epirubicin and epirubicinol was good but epirubicinol peak shape poor 4 70:13:17 C18, 5 μ, 150 mm at 40°C CP, epirubicin and epirubicinol sharp peak and good separation Sl. No. Mobile phase (water at pH 4.0: MeOH: ACN) Column Result 1 70:5:25 C18, 5 μ, 150 mm at RT CP separated out, but merged peaks of epirubicin and epirubicinol 2 70:10:20 C18, 5 μ, 150 mm at RT CP separated out, but the separation of epirubicin and epirubicinol was poor 3 70:13:17 C18, 5 μ, 150 mm at RT CP separated out, but the separation of epirubicin and epirubicinol was good but epirubicinol peak shape poor 4 70:13:17 C18, 5 μ, 150 mm at 40°C CP, epirubicin and epirubicinol sharp peak and good separation Chromatographic method validation To begin with, we assessed the reliability of chromatographic method to detect and quantitate FU, DHFU, FUDR, FUR, EP, EPol and CP. The method was performed as described in the previous section. The peaks of the compounds were picked by comparing with the blank profile. RT (mean ± SD) were as follows: for DHFU (λmax 200 nm) tr = 7.74 ± 0.019 min, FU (λmax 265 nm) tr = 10.51 ± 0.033 min, FUR (λmax 270 nm) tr = 22.66 ± 0.106, FUDR (λmax 270 nm) tr = 32.35 ± 0.109 min and EPol (λmax 254 nm) tr = 38.96 ± 0.087, EP (λmax 254 nm) tr = 40.81 ± 0.142 min, CP (λmax 195 nm) tr = 60.31 ± 0.069 (Figure 2). Calibration curve for standard solutions was plotted over the ranges stated in the previous section (Supplementary Figure S4). The regression equations were y = 6.697x + 2.070 (r2 = 0.9909 ± 0.0054) for FU, y = 3.092x + 4.038 (r2 = 0.9923 ± 0.0034) for DHFU, y = 1.187x – 5.127 (r2 = 0.9913 ± 0.0027) for FUDR, y = 0.7032 × – 0.5587 (r2 = 0.9918 ± 0.0039) for FUR, y = 0.1055 × – 0.0784 (r2 = 0.9963 ± 0.0002) for EP, y = 0.1078x – 0.0089 (r2 = 0.9880 ± 0.0009) for EPol, y = 0.0690x – 1.008 (r2 = 0.9910 ± 0.0045) for CP, respectively. For all the compounds, the coefficients of determination (r2 values) prove that the method was linear in the specified range for all the compounds tested. The results obtained for the calibration curve and the r2 values for the individual compounds are summarized in Table III. Figure 2. View largeDownload slide Chromatograms of blank and standard, respectively, of DHFU 30 μg/mL (a and b at 200 nm), FU 6 μg/mL (c and d at 265 nm), FUR 15 μg/mL and FUDR 30 μg/mL (e and f at 270 nm), EPol and EP both 30 μg/mL (g and h at 254 nm), CP 300 μg/mL (i and j at 195 nm). Figure 2. View largeDownload slide Chromatograms of blank and standard, respectively, of DHFU 30 μg/mL (a and b at 200 nm), FU 6 μg/mL (c and d at 265 nm), FUR 15 μg/mL and FUDR 30 μg/mL (e and f at 270 nm), EPol and EP both 30 μg/mL (g and h at 254 nm), CP 300 μg/mL (i and j at 195 nm). Table III. The retention times (RT) and other parameters of validation (linearity, LOD, LOQ, precision and accuracy) of the drugs and their metabolites in water (pH 4.0) Sl no. Compound RT (mean ± SD) LOD (ng/ml) LOQ (ng/ml) r2 Value (mean ± SD) Precision (RSD %) Accuracy (%) 1 FU 10.51 ± 0.033 2 10 0.9909 ± 0.0054 1.60 100.07 2 FUR 22.66 ± 0.106 30 150 0.9918 ± 0.0039 1.45 98.91 3 FUDR 32.35 ± 0.109 20 100 0.9913 ± 0.0027 0.53 99.47 4 DHFU 7.74 ± 0.019 10 50 0.9923 ± 0.0034 1.52 100.10 5 EP 40.81 ± 0.142 30 150 0.9963 ± 0.0002 0.71 99.93 6 EPol 38.96 ± 0.087 10 50 0.9880 ± 0.0009 1.31 99.28 7 CP 60.31 ± 0.069 200 1000 0.9910 ± 0.0045 0.27 99.85 Sl no. Compound RT (mean ± SD) LOD (ng/ml) LOQ (ng/ml) r2 Value (mean ± SD) Precision (RSD %) Accuracy (%) 1 FU 10.51 ± 0.033 2 10 0.9909 ± 0.0054 1.60 100.07 2 FUR 22.66 ± 0.106 30 150 0.9918 ± 0.0039 1.45 98.91 3 FUDR 32.35 ± 0.109 20 100 0.9913 ± 0.0027 0.53 99.47 4 DHFU 7.74 ± 0.019 10 50 0.9923 ± 0.0034 1.52 100.10 5 EP 40.81 ± 0.142 30 150 0.9963 ± 0.0002 0.71 99.93 6 EPol 38.96 ± 0.087 10 50 0.9880 ± 0.0009 1.31 99.28 7 CP 60.31 ± 0.069 200 1000 0.9910 ± 0.0045 0.27 99.85 Table III. The retention times (RT) and other parameters of validation (linearity, LOD, LOQ, precision and accuracy) of the drugs and their metabolites in water (pH 4.0) Sl no. Compound RT (mean ± SD) LOD (ng/ml) LOQ (ng/ml) r2 Value (mean ± SD) Precision (RSD %) Accuracy (%) 1 FU 10.51 ± 0.033 2 10 0.9909 ± 0.0054 1.60 100.07 2 FUR 22.66 ± 0.106 30 150 0.9918 ± 0.0039 1.45 98.91 3 FUDR 32.35 ± 0.109 20 100 0.9913 ± 0.0027 0.53 99.47 4 DHFU 7.74 ± 0.019 10 50 0.9923 ± 0.0034 1.52 100.10 5 EP 40.81 ± 0.142 30 150 0.9963 ± 0.0002 0.71 99.93 6 EPol 38.96 ± 0.087 10 50 0.9880 ± 0.0009 1.31 99.28 7 CP 60.31 ± 0.069 200 1000 0.9910 ± 0.0045 0.27 99.85 Sl no. Compound RT (mean ± SD) LOD (ng/ml) LOQ (ng/ml) r2 Value (mean ± SD) Precision (RSD %) Accuracy (%) 1 FU 10.51 ± 0.033 2 10 0.9909 ± 0.0054 1.60 100.07 2 FUR 22.66 ± 0.106 30 150 0.9918 ± 0.0039 1.45 98.91 3 FUDR 32.35 ± 0.109 20 100 0.9913 ± 0.0027 0.53 99.47 4 DHFU 7.74 ± 0.019 10 50 0.9923 ± 0.0034 1.52 100.10 5 EP 40.81 ± 0.142 30 150 0.9963 ± 0.0002 0.71 99.93 6 EPol 38.96 ± 0.087 10 50 0.9880 ± 0.0009 1.31 99.28 7 CP 60.31 ± 0.069 200 1000 0.9910 ± 0.0045 0.27 99.85 Drug and drug metabolite analysis in plasma: method validation Various methods for the recovery of drugs from plasma had been screened using organic solvents and solid phase extraction. For instance, solid phase extraction from plasma or diluted plasma and elution in acetonitrile, or solid phase extraction from plasma and elution with water pH 4.0, methanol and acetonitrile—70:13:17 v/v/v, or solid phase extraction followed by elution in methanol, or extraction using TCA were assessed, the details of extraction and the drawbacks observed are tabulated in Supplementary Table 1. Finally, the current method, acetonitrile-based precipitation was found to be appropriate since it yielded less interfering peaks with increased recovery. The chromatograms of the compounds recovered from plasma as against their unspiked blanks are given in Figure 3. Figure 3. View largeDownload slide Chromatograms of unspiked and spiked plasma, respectively, of DHFU 30 μg/mL (a and b at 200 nm), FU 6 μg/mL (c and d at 265 nm), FUR 15 μg/mL and FUDR 30 μg/mL (e and f at 270 nm), EPol and EP both 30 μg/mL (g and h at 254 nm), CP 300 μg/mL (i and j at 195 nm). Figure 3. View largeDownload slide Chromatograms of unspiked and spiked plasma, respectively, of DHFU 30 μg/mL (a and b at 200 nm), FU 6 μg/mL (c and d at 265 nm), FUR 15 μg/mL and FUDR 30 μg/mL (e and f at 270 nm), EPol and EP both 30 μg/mL (g and h at 254 nm), CP 300 μg/mL (i and j at 195 nm). Linearity and recovery Standard curves for all the seven compounds were produced with known concentrations spiked in plasma FU (range 0.1–10 μg/mL); DHFU, FUDR and EP (range 0.1–50 μg/mL); EPol (range 1–50 μg/mL), FUR (range 5–200 μg/mL); and CP (range 10–500 μg/mL). Seven points were plotted with AUC values of these drugs against their respective concentrations and linear regression analysis performed on the resultant curves (Supplementary Figure S5). The regression equations were y = 7.0950 × –0.3059 (r2 = 0.9993 ± 0.0062) for FU, y = 1.5460 × –0.5348 (r2 = 0.9997 ± 0.0073) for DHFU, y = 2.0350 × + 1.7980 (r2 = 0.9970 ± 0.003) for FUDR, y = 0.9183 × + 2.1740 (r2 = 0.9998 ± 0.0034) for FUR, y = 0.0670 ×–0.1514 (r2 = 0.9820 ± 0.0004) for EP, y = 0.2251 × –0.2488 (r2 = 0.9985 ± 0.0006) for EPol and y = 0.0547 × + 0.4741 (r2 = 0.9977 ± 0.0029) for CP, respectively. For all the compounds, the coefficients of determination (r2 values) prove that the method was linear in the specified range for all the compounds tested. The results obtained for the calibration curve and the r2 values for the individual compounds are summarized in Table IV. The percentage recovery was calculated by comparing the drug concentration from spiked plasma samples and that from standard solutions, as determined from peak areas, FU and metabolites had a recovery of >93%, whereas that of EP, EPol and CP were >78%. The percentage recoveries for each compound from plasma are as given in Table IV. Table IV. The retention times (RT), linearity (r2 values) and percentage recovery of the drugs and their metabolites in matrix Sl no. Compound RT (mean ± SD) r2 Value (mean ± SD) % Recovery Precision (RSD %) Accuracy (%) 1 FU 10.51 ± 0.033 0.9993 ± 0.0062 99.79 1.61 98.9 2 FUR 22.66 ± 0.106 0.9998 ± 0.0034 93.56 1.48 99.06 3 FUDR 32.35 ± 0.109 0.9970 ± 0.003 98.93 1.79 98.26 4 DHFU 7.74 ± 0.019 0.9997 ± 0.0073 98.58 1.64 101.72 5 EP 40.81 ± 0.142 0.9820 ± 0.0004 78.9 1.18 99.77 6 EPol 38.96 ± 0.087 0.9985 ± 0.0006 78.35 0.54 99.75 7 CP 60.31 ± 0.069 0.9977 ± 0.0029 79.34 0.5 99.62 Sl no. Compound RT (mean ± SD) r2 Value (mean ± SD) % Recovery Precision (RSD %) Accuracy (%) 1 FU 10.51 ± 0.033 0.9993 ± 0.0062 99.79 1.61 98.9 2 FUR 22.66 ± 0.106 0.9998 ± 0.0034 93.56 1.48 99.06 3 FUDR 32.35 ± 0.109 0.9970 ± 0.003 98.93 1.79 98.26 4 DHFU 7.74 ± 0.019 0.9997 ± 0.0073 98.58 1.64 101.72 5 EP 40.81 ± 0.142 0.9820 ± 0.0004 78.9 1.18 99.77 6 EPol 38.96 ± 0.087 0.9985 ± 0.0006 78.35 0.54 99.75 7 CP 60.31 ± 0.069 0.9977 ± 0.0029 79.34 0.5 99.62 Table IV. The retention times (RT), linearity (r2 values) and percentage recovery of the drugs and their metabolites in matrix Sl no. Compound RT (mean ± SD) r2 Value (mean ± SD) % Recovery Precision (RSD %) Accuracy (%) 1 FU 10.51 ± 0.033 0.9993 ± 0.0062 99.79 1.61 98.9 2 FUR 22.66 ± 0.106 0.9998 ± 0.0034 93.56 1.48 99.06 3 FUDR 32.35 ± 0.109 0.9970 ± 0.003 98.93 1.79 98.26 4 DHFU 7.74 ± 0.019 0.9997 ± 0.0073 98.58 1.64 101.72 5 EP 40.81 ± 0.142 0.9820 ± 0.0004 78.9 1.18 99.77 6 EPol 38.96 ± 0.087 0.9985 ± 0.0006 78.35 0.54 99.75 7 CP 60.31 ± 0.069 0.9977 ± 0.0029 79.34 0.5 99.62 Sl no. Compound RT (mean ± SD) r2 Value (mean ± SD) % Recovery Precision (RSD %) Accuracy (%) 1 FU 10.51 ± 0.033 0.9993 ± 0.0062 99.79 1.61 98.9 2 FUR 22.66 ± 0.106 0.9998 ± 0.0034 93.56 1.48 99.06 3 FUDR 32.35 ± 0.109 0.9970 ± 0.003 98.93 1.79 98.26 4 DHFU 7.74 ± 0.019 0.9997 ± 0.0073 98.58 1.64 101.72 5 EP 40.81 ± 0.142 0.9820 ± 0.0004 78.9 1.18 99.77 6 EPol 38.96 ± 0.087 0.9985 ± 0.0006 78.35 0.54 99.75 7 CP 60.31 ± 0.069 0.9977 ± 0.0029 79.34 0.5 99.62 LOD and LOQ The LOD and LOQ of the compounds were studied in water at pH 4.0. The LOD and LOQ concentrations of the compounds are given in Table III. Our method was able to detect all the compounds in the nanogram/milliliter range except in CP where LOD was 0.2 μg/mL and LOQ was 1 μg/mL. Precision and accuracy The precision and accuracy were calculated as mentioned in the previous section for the compounds in both water (pH 4.0) and plasma. Precision (%RSD) was <2% for all compounds, both in water (pH 4.0) and plasma. Similarly, accuracy was >98% for all compounds (Tables III and IV). Stability The stability of each of these compounds in water at pH 4.0 was studied by analyzing at various time points over a period of 24 h. FU, DHFU and EP were the most stable among the compounds studied at room temperature, with a difference in AUC measured after 24 h, not more than 0.5 mAU*min. CP, EPol and FUDR were initially stable but the AUC values decreased thereafter. For instance, AUC of CP decreased from 23.62 mAU*min at 5 h to 6.10 mAU*min at 24 h, for EPol tailing of the peak was observed with decrease in AUC from 3.41 mAU*min to 1.85 mAU*min and for FUDR, the initial peak with AUC 37.68 mAU*min was not observed after 24 h. FUR was found to be stable for 3 h, with a sharp dip in AUC assessed at the end of 5 h (9.51–1.39 mAU*min) (Figure 4a and b). In total, the analysis indicated that stability of FU, DHFU and EP was higher followed by CP, EPol, FUDR and FUR. Figure 4. View largeDownload slide (a) Stability of FU, DHFU, FUR and FUDR in water (pH 4.0). Plotted time in hours (h) on the x-axis and area under curve (AUC) in mAU*mins on the x-axis and (b) stability of EP, EPol and CP in water (pH 4.0). Plotted time in hours (h) on the x-axis and area under curve (AUC) in mAU*min on the y-axis. Figure 4. View largeDownload slide (a) Stability of FU, DHFU, FUR and FUDR in water (pH 4.0). Plotted time in hours (h) on the x-axis and area under curve (AUC) in mAU*mins on the x-axis and (b) stability of EP, EPol and CP in water (pH 4.0). Plotted time in hours (h) on the x-axis and area under curve (AUC) in mAU*min on the y-axis. Validation of the peaks by mass spectrometry The UV absorption peaks corresponding to each of the compounds were confirmed using mass spectrometry. The fractions collected upon HPLC separation was analyzed in MS as described above. The m/z values thus obtained was compared with that of the pure compounds. FU, FUR and FUDR were measured in the negative mode as [M-H]− with m/z ratios, 128.90, 260.75 and 244.90, respectively. DHFU, EP, EPol and CP were measured in the positive mode as [M + H]+ with m/z ratios 132.86, 544.02 and 546.10, respectively (Figure 5). The mass tolerance for the measurement was set at ±0.5 Da. The m/z values estimated for the HPLC peak fraction coincided with the observed masses of the standard compounds confirming the identity of compounds eluting in the corresponding peaks (Table V). Figure 5. View largeDownload slide Validation of UV-HPLC peaks of FEC and their metabolites, namely, DHFU (a), FU (b), FUR (c), FUDR (d), EP (e), EPol (f) and CP (g). FU, FUR and FUDR were measured in the negative mode and DHFU, EP, EPol and CP in the positive mode (Cut-off ±0.5 Da). Figure 5. View largeDownload slide Validation of UV-HPLC peaks of FEC and their metabolites, namely, DHFU (a), FU (b), FUR (c), FUDR (d), EP (e), EPol (f) and CP (g). FU, FUR and FUDR were measured in the negative mode and DHFU, EP, EPol and CP in the positive mode (Cut-off ±0.5 Da). Table V. Validation of UV-HPLC peaks of FEC and their metabolites by MS analysis Sl no. Compound RT min (mean ± SD) ESI Mode Expected mass (m/z) Observed mass (peak fraction) (m/z) (±0.5 Da) 1 FU 10.51 ± 0.033 − 129.01 [M−H]− 128.90 [M−H]− 2 DHFU 7.74 ± 0.019 + 133.04 [M+H]+ 132.86 [M+H]+ 3 FUR 32.35 ± 0.109 − 261.06 [M−H]− 260.75 [M−H]− 4 FUDR 22.66 ± 0.106 − 245.06 [M−H]− 244.90 [M−H]− 5 EP 40.81 ± 0.142 + 544.18 [M+H]+ 544.02 [M+H]+ 6 EPol 38.96 ± 0.087 + 546.19 [M+H]+ 546.10 [M+H]+ 7 CP 60.31 ± 0.069 + 261.03 [M+H]+ 260.71 [M+H]+ Sl no. Compound RT min (mean ± SD) ESI Mode Expected mass (m/z) Observed mass (peak fraction) (m/z) (±0.5 Da) 1 FU 10.51 ± 0.033 − 129.01 [M−H]− 128.90 [M−H]− 2 DHFU 7.74 ± 0.019 + 133.04 [M+H]+ 132.86 [M+H]+ 3 FUR 32.35 ± 0.109 − 261.06 [M−H]− 260.75 [M−H]− 4 FUDR 22.66 ± 0.106 − 245.06 [M−H]− 244.90 [M−H]− 5 EP 40.81 ± 0.142 + 544.18 [M+H]+ 544.02 [M+H]+ 6 EPol 38.96 ± 0.087 + 546.19 [M+H]+ 546.10 [M+H]+ 7 CP 60.31 ± 0.069 + 261.03 [M+H]+ 260.71 [M+H]+ Table V. Validation of UV-HPLC peaks of FEC and their metabolites by MS analysis Sl no. Compound RT min (mean ± SD) ESI Mode Expected mass (m/z) Observed mass (peak fraction) (m/z) (±0.5 Da) 1 FU 10.51 ± 0.033 − 129.01 [M−H]− 128.90 [M−H]− 2 DHFU 7.74 ± 0.019 + 133.04 [M+H]+ 132.86 [M+H]+ 3 FUR 32.35 ± 0.109 − 261.06 [M−H]− 260.75 [M−H]− 4 FUDR 22.66 ± 0.106 − 245.06 [M−H]− 244.90 [M−H]− 5 EP 40.81 ± 0.142 + 544.18 [M+H]+ 544.02 [M+H]+ 6 EPol 38.96 ± 0.087 + 546.19 [M+H]+ 546.10 [M+H]+ 7 CP 60.31 ± 0.069 + 261.03 [M+H]+ 260.71 [M+H]+ Sl no. Compound RT min (mean ± SD) ESI Mode Expected mass (m/z) Observed mass (peak fraction) (m/z) (±0.5 Da) 1 FU 10.51 ± 0.033 − 129.01 [M−H]− 128.90 [M−H]− 2 DHFU 7.74 ± 0.019 + 133.04 [M+H]+ 132.86 [M+H]+ 3 FUR 32.35 ± 0.109 − 261.06 [M−H]− 260.75 [M−H]− 4 FUDR 22.66 ± 0.106 − 245.06 [M−H]− 244.90 [M−H]− 5 EP 40.81 ± 0.142 + 544.18 [M+H]+ 544.02 [M+H]+ 6 EPol 38.96 ± 0.087 + 546.19 [M+H]+ 546.10 [M+H]+ 7 CP 60.31 ± 0.069 + 261.03 [M+H]+ 260.71 [M+H]+ Stability of the compounds in plasma stored at −80°C In order to assess the effects of sample storage on recovery of the parent drugs and metabolites from plasma, the spiked plasma samples were incubated for various time points over a period of 72 h, and analyzed in triplicates as described in the previous section. Three concentrations of the respective analyte ranging from low, medium and high levels were tested. There were no statistically significant differences in the concentration of the compounds (FU, DHFU, FUDR, EPol and CP) analyzed upon storage at −80°C for the given time periods. However, in the case of FUR and EP, values indicated a slight but statistically significant (P-value <0.05) decrease in the recovery at the higher concentration level at 72 h (Figure 6). Figure 6. View largeDownload slide Recovery of FU, DHFU, FUR, FUDR, EP, EPol and CP from matrix on storage at −80oC after 24 , 48 and 72 h. The samples were spiked with three concentrations of each drug (a–g) and concentration (μg/mL) was plotted against time (h). The values of FUR and EP showed a significant difference at higher concentration after 72 h (*P < 0.05). Figure 6. View largeDownload slide Recovery of FU, DHFU, FUR, FUDR, EP, EPol and CP from matrix on storage at −80oC after 24 , 48 and 72 h. The samples were spiked with three concentrations of each drug (a–g) and concentration (μg/mL) was plotted against time (h). The values of FUR and EP showed a significant difference at higher concentration after 72 h (*P < 0.05). Patient samples The analysis was performed in patient samples to assess the clinical applicability of our method. The samples from patients were collected after 3 h of FU and EP injections and immediately after CP injection at the first cycle of the protocol. The samples were analyzed immediately following isolation to avoid degradation as indicated from the stability analysis. Our method was able to detect the drugs and their metabolites in plasma of patients. FU was detected in all the samples (range 0.2–0.9 μg/mL), CP (range 66.1–159.7 μg/mL) and DHFU (range 0.30–1.2 μg/mL) were detected in all but one samples, FUDR (range 1.3–3.0 μg/mL) was seen in three and EP (0.6 and 0.2 μg/mL) in two samples. EPol and FUR were seen only in one of the samples tested (1.9 and 146.3 μg/mL, respectively) (Table VI). Table VI. The estimation of compounds (FU, EP—their metabolites and CP) in the plasma of patients undergoing chemotherapy with FEC regimen. Patient (↓) Dosage FU (μg/ml) DHFU (μg/ml) FUR (μg/ml) FUDR (μg/ml) EP (μg/ml) EPol (μg/ml) CP (μg/ml) 1 FEC-90 0.630 0.711 145.6 3.048 12.772 9.204 154.949 2 FEC-60 0.583 1.424 ND ND ND ND 132.729 3 FEC-90 0.276 1.543 ND 1.302 2.891 ND 115.755 4 FEC-60 0.336 ND ND ND ND ND 76.693 5 FEC-90 0.252 0.938 ND 1.650 ND ND ND 6 FEC-90 0.268 0.658 ND ND ND ND 95.614 Patient (↓) Dosage FU (μg/ml) DHFU (μg/ml) FUR (μg/ml) FUDR (μg/ml) EP (μg/ml) EPol (μg/ml) CP (μg/ml) 1 FEC-90 0.630 0.711 145.6 3.048 12.772 9.204 154.949 2 FEC-60 0.583 1.424 ND ND ND ND 132.729 3 FEC-90 0.276 1.543 ND 1.302 2.891 ND 115.755 4 FEC-60 0.336 ND ND ND ND ND 76.693 5 FEC-90 0.252 0.938 ND 1.650 ND ND ND 6 FEC-90 0.268 0.658 ND ND ND ND 95.614 ND—not detected. Table VI. The estimation of compounds (FU, EP—their metabolites and CP) in the plasma of patients undergoing chemotherapy with FEC regimen. Patient (↓) Dosage FU (μg/ml) DHFU (μg/ml) FUR (μg/ml) FUDR (μg/ml) EP (μg/ml) EPol (μg/ml) CP (μg/ml) 1 FEC-90 0.630 0.711 145.6 3.048 12.772 9.204 154.949 2 FEC-60 0.583 1.424 ND ND ND ND 132.729 3 FEC-90 0.276 1.543 ND 1.302 2.891 ND 115.755 4 FEC-60 0.336 ND ND ND ND ND 76.693 5 FEC-90 0.252 0.938 ND 1.650 ND ND ND 6 FEC-90 0.268 0.658 ND ND ND ND 95.614 Patient (↓) Dosage FU (μg/ml) DHFU (μg/ml) FUR (μg/ml) FUDR (μg/ml) EP (μg/ml) EPol (μg/ml) CP (μg/ml) 1 FEC-90 0.630 0.711 145.6 3.048 12.772 9.204 154.949 2 FEC-60 0.583 1.424 ND ND ND ND 132.729 3 FEC-90 0.276 1.543 ND 1.302 2.891 ND 115.755 4 FEC-60 0.336 ND ND ND ND ND 76.693 5 FEC-90 0.252 0.938 ND 1.650 ND ND ND 6 FEC-90 0.268 0.658 ND ND ND ND 95.614 ND—not detected. Discussion A study of the literature on the various methodologies developed revealed that there are HPLC-UV-based methods for the estimation of above-mentioned drugs and their metabolites either individually or as a combination in mixtures. Despite the fact that methods are available that combine FU and CP using HPLC-UV, the estimation of EP in most cases has been through a fluorescence detector (8, 14–17), and we were unable to identify a method that is suitable for the quantification of the triple-drug combination (FEC) and their metabolites simultaneously in plasma based on HPLC-UV (Supplementary Tables 2–4). In the case of FU the drug was estimated using a combination of gas chromatography–mass Spectrometry (18). In addition, metabolites of FU dihydrofluorouracil (DHFU), α-fluoro-β-ureidopropionic acid and α-fluoro-β-alanine (FBAL) were also estimated using gas chromatography–mass spectrometry (19). Studies had revealed the advantages of liquid chromatography based estimation of FU over gas chromatography, using 50 mM phosphate buffer at pH 3.0 as mobile phase and in addition also presented a tedious procedure for the extraction of drug from plasma (17, 20). A subsequent HPLC-UV method for the estimation of FU used a mobile phase comprising combination phosphate buffer and 8% methanol in phosphate buffer, pH 5.5 in a single run. Plasma extraction was by saturating with ammonium sulfate followed by n-propanol and ether-based extraction and the LOD of this method was 0.1 μg/mL (21). Similarly, studies have used a varying combination of mobile phases comprising phosphate buffer (3, 22), potassium phosphate–methanol (23), potassium dihydrogen phosphate and methanol (24), tetrabutylammonium hydrogen sulfate and potassium phosphate buffer (25). A summary of various mobile phases, detection wavelength and LOD and LOQ values reported for FU and metabolites are presented in Supplementary Table 2. In addition, fluorescence-based detection method was developed for FU and FUDR from the plasma of patients after derivatization using 3-bromomethyl-6, 7-dimethoxy-l-methyl-2(1H)-quinoxalinone (Br-DMEQ). The mobile phase used was simple, 35% methanol in water and the fluorescence was measured at excitation and emission wavelengths of 370 and 455 nm, respectively (26). Enzyme-based estimation have also been employed for instance FdUMP, an active antineoplastic metabolite of FU from tissues was measured based on the stoichiometric inhibition of Lactobacillus casei TS and was sensitive in detecting as low as 1 pmol of FdUMP (27). FU and the three metabolites DHFU, FUR and FUDR have been estimated using UV-HPLC method but their LOQ was much higher and in the μg/mL range. The LOQ was 0.5 μg/mL for FU, 1 μg/mL for DHFU, 3 μg/mL for FUR and FUDR. The relatively poor LOQ and recovery of this method could be attributed to the choice of detection wavelength. The study had chosen 210 nm for all the compounds, however, FU, FUR and FUDR have a maximum absorption in the range of 254–280 nm whereas DHFU has around 190 nm and hence, a compromise had to be made in case of having system that could detect only one wavelength (28) (Supplementary Table 2). Most of the studies on EP and EPol have been based on fluorescence detection at λex470–480 nm and λem550–560 nm (9, 29, 30) (Supplementary Table 3). Electron spray mass spectrometry has also been employed in the detection of EP, on elution with ammonium formate and acetonitrile at acidic pH of 3.0 (31). Very few studies are available for UV-HPLC method for estimation of EP, of which one of them used sodium formate, methanol and acetonitrile as mobile phase and detection wavelength was set at 280 nm (32). Another study used methanol and water as mobile phase and detection was at 254 nm. The LOD was 40 ng/mL, which is higher than that of our method (33). Cyclophosphamide and its metabolite have been estimated using methanol: 20 mM ammonium acetate containing 1% acetic acid (30:70 v/v) as mobile phase, and detection was mass spectrometry-based (34). Assay of cyclophosphamide in bulk drug used mobile phase of 10% acetonitrile as Solvent A and 70% acetonitrile as Solvent B and gradient was used for resolving the compound and the UV detection wavelength was set at 195 nm (10) (Supplementary Table 4). There are studies available in the literature that studied estimation of multiple drug combination by a UV-HPLC method (Supplementary Table 5). For instance, a study measured the three drugs 5-fluorouracil, adriamycin and cyclophosphamide with a mobile phase of 0.05 M disodium hydrogen phosphate and acetonitrile (65:35 v/v) at pH 3.7 and Diode Array detector set at 266 nm, 254 nm and 195 nm (17). FU and CP have also been measured in combination as part of the five antineoplastic agents using a mobile phase comprising water (pH 4.0), methanol, acetonitrile (70:13:17 v/v/v) and detection using DAD (16). Another plasma-based method for the estimation of FU and CP used the same mobile phase at the ratio 68:13:19 v/v/v (35). The LOD and LOQ of these methods ranged from 0.07–0.1 μg/mL and 0.098–1.0 μg/mL, respectively, for FU, whereas LOD and LOQ of our study was 2 and 10 ng/mL, respectively. The LOD and LOQ of CP were 0.1–0.3 and 0.15–0.5 μg/mL, respectively, lower than that in our study, 0.2 and 1 μg/mL, respectively. However, it must be noted that the multidrug estimation methodologies did not involve the drug EP in the mixture, hence a method was developed to estimate FEC triple-drug combination. In our estimations of drug levels in patient samples who were treated with FEC protocols, FEC-60 and FEC-90 where the dosage of FU and CP is the same with the difference being in the dose of EP, we observed that some drugs are observed at detectable levels while others are not. This variability in detection could be due to the influence of dose, genetic/clinico-pathological features, for instance, EP was observed in two patients from a total six and at a higher level in a FEC-90 relative to FEC-60. To understand the differences in observed values would require a larger series of samples to assess for the correlation of dose and genetic/clinico-pathological features with drug levels. 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Google Scholar CrossRef Search ADS © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Published: Mar 28, 2018

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