Simultaneous HPLC Determination of Cisatracurium and Propofol in Human Plasma via Fluorometric Detection

Simultaneous HPLC Determination of Cisatracurium and Propofol in Human Plasma via Fluorometric... Abstract The proposed method describes a high performance liquid chromatographic method with fluoremetric detection for the determination of cisatracurium (CIS) and propofol (PRP) simultaneously, which are co-administered as a pre-operative injection mixture. The separation of the two drugs was achieved using monolithic column (100 mm and 4.6 mm internal diameter) and mixture of methanol and 0.1 M phosphate buffer in the ratio of 80:20 (v/v) at pH 4.5 as a mobile phase. The fluorescence detection was carried out at 230/324 nm. The procedure showed good linearity through the concentration ranges of 0.01–1.00 μg/mL and 0.1–3.0 μg/mL with limits of detection of 0.002, 0.030 μg/mL and limits of quantification of 0.006, 0.100 μg/mL for CIS and PRP, respectively. Simultaneous determination of CIS and PRP in spiked human plasma samples was additionally executed and the results were satisfactory precise and accurate. Introduction Cisatracurium (CIS, Figure 1a) is a competitive neuromuscular blocker that acts by competing with acetylcholine for receptors on the motor end-plate of the neuromuscular junctions to produce blockade. Restoration of normal neuromuscular function can be hastened by increasing the concentration of acetylcholine at the motor end-plate by giving an anticholinesterase such as neostigmine (1). Chemically it is (1 R,1′R,R,2′R)-2,2-[1,5-pentanediylbis-[oxy(3-oxo-3,1-propanediyl)]]-bis[1-[(3,4-dimethoxyphenyl)-methyl]-1,2,3,4-tetrahydro-6,7–dimethoxy-2-methylisoquinolin-ium] dibenzene sulfonate (2). Regarding official methods; the USP monograph (2), and the BP monograph (3), suggested LC method with UV detection at 280 nm for estimation of CIS raw material. The literature revealed limited methods for CIS determination as HPLC (4–8), spectrofluorimetry (9), voltammetry (10) and capillary electrophoresis (11). Figure 1. View largeDownload slide The structural formulae for the studied drugs. Figure 1. View largeDownload slide The structural formulae for the studied drugs. Propofol (PRP, Figure 1b) is 2,6-Bis(1-methylethyl)phenol (2). It acts as a short-acting anesthetic given intravenously to induce and also maintain general anesthesia. It is prescribed for sedation in adult patients undergoing diagnostic procedures, in those undergoing surgery with local or regional anesthesia, and in ventilated adult patients under intensive care. When used for anesthesia, induction is rapid, as is recovery. Propofol has no analgesic effect, so supplementary analgesia may be used (1). The USP recommended gas chromatography equipped with a flame-ionization detector (2) and a LC method was mentioned in the BP for PRP assay (3). Previous publications for the determination of PRP have been reported such as; spectrophotometric analysis (12), sequential injection analysis (13), HPLC (4, 14–28) and electrochemical methods (29). Reviewing the literature indicating; only one research was found in the literature for the analysis of the CIS and PRP using HPLC with UV detection and MS for the identification of the degradation products (4). Combination of CIS and PRP is commonly used during surgery as a pre-operative procedure (30), as CIS besylate is a neuromuscular blocking agent, used as an adjunct to general anesthesia (1). Propofol on the other hand is a general anesthetic, prescribed for induction and maintenance of general anesthesia. Co-administration of the two drugs during surgery is frequent, so our aim was developing a reliable method with reaching low detection limit that permits their determination in human plasma for the purpose of therapeutic drug monitoring. HPLC coupled with fluorescence detection proved to be a reliable technique in such cases. Since the two drugs should be observed together after anesthesia, the method can be adopted for their determination in spiked human plasma. Experimental Equipment HPLC experiments were done using a Shimadzu LC-20AD Chromatograph (Japan) equipped with a Rheodyne injector valve and a 20 μL loop and a RF-10AXL fluorescence detector. A Consort NV P-901 pH –Meter (Belgium) was used for pH measurements. Materials and reagents - CIS with a purity of 99.2% was provided by National Organization for Drug Control and Research (NODCAR), Cairo, Egypt. (The purity was specified by the organization) - Propofol with a purity of 99.90% (as stated by the manufacture) was also kindly provided by NODCAR, Cairo, Egypt. - CIS ampoules, batch # 15023, labeled to contain 25.0 mg /2.5 mL, (product of Hameln Pharmaceutical Germany, for Sunny Medical Group, Cairo, Egypt). - Propofol ampoules, batch # 250127, labeled to contain 10 mg/1 ml, (product of Astrazeneca Pharmaceutical Company, Cairo, Egypt). - Methanol, acetonitrile and n-propanol (HPLC grade) were purchased from Sigma-Aldrich (Germany). - Sodium hydroxide and sodium dihydrogen phosphate were purchased from ADWIC. (Cairo, Egypt). - Orthophosphoric acid (85%, w/v) was purchased from Riedel-deHäen (Sleeze, Germany). - Plasma samples were obtained from Blood-Bank, Mansoura University Hospital, Mansoura, Egypt and were kept frozen until use after gentle thawing. Chromatographic conditions The column used for separation was Chromolith Reversed Phase Monolithic column (100 mm × 4.6 mm i.d.), Merck, Germany. Mobile phase used containing methanol and 0.1 M orthophosphoric acid buffer in the ratio of 80:20, v/v respectively at pH 4.5 was used. Detection wavelengths were 230 nm (excitation) and 324 nm (emission). The separation was done using flow rate 1 mL/min. Standard solutions Stock solutions were prepared by dissolving 1.0 mg/mL of either CIS or PRP in methanol, and completing the volume to 100 mL in a volumetric flask. General recommended procedures Calibration graphs construction Aliquots were accurately transferred to 10 mL volumetric flasks, completed to volume with the mobile phase. The final concentrations would be in the range of 0.01–1.0 μg/mL for CIS and 0.1-3.0 μg/mL for PRP. The average peak areas were plotted against the final concentration of the drug in μg/mL to get the calibration graphs. Then, the regression equations were derived. Analysis of CIS/PRP synthetic mixtures Mixtures of variable ratios of CIS and PRP working standard solutions were transferred into a set of 10 mL volumetric flasks. The procedure described under “Construction of calibration graphs” was carried out. By referring to the calibration graphs; the percentage recovery for each concentration of the drugs was calculated. Analysis of the studied drugs in their ampoules Analysis of CIS in ampoules Five CIS ampoules were emptied and mixed. An accurately measured volume equivalent to 25.0 mg of CIS was transferred into a clean dry 100 mL volumetric flask. The flask was completed with methanol to the mark. Dilution with methanol was done to obtain working solutions within the studied concentration range. The general procedure described under “Construction of Calibration Graphs” was performed. The content of ampoules was then calculated referring to the regression equation. Analysis of PRP in ampoules A volume from PRP ampoules containing 10.0 mg of PRP was transferred into 100 mL volumetric flask, and then the same steps for CIS ampoules were followed (Figure 2). Figure 2. View largeDownload slide Typical chromatogram of synthetic mixture under the described chromatographic conditions: (A): CIS (0.1 μg/mL). (B): PRP (0.5 μg/mL). Figure 2. View largeDownload slide Typical chromatogram of synthetic mixture under the described chromatographic conditions: (A): CIS (0.1 μg/mL). (B): PRP (0.5 μg/mL). Figure 3 shows the chromatogram for CIS and PRP ampoules using the described method. Figure 3. View largeDownload slide Typical chromatogram of ampoules of the studied drugs under the described chromatographic conditions: (A): CIS (0.1 μg/mL). (B): PRP (0.5 μg/mL). Figure 3. View largeDownload slide Typical chromatogram of ampoules of the studied drugs under the described chromatographic conditions: (A): CIS (0.1 μg/mL). (B): PRP (0.5 μg/mL). Analysis of drugs in human plasma Construction of calibration graphs in spiked human plasma Standard calibration graphs were constructed for each of CIS and PRP in human plasma in the ranges of (0.01–0.05 μg/mL for CIS and 0.1–0.5 μg/mL for PRP) by adding appropriate volumes of CIS and PRP working solutions into plasma samples. The calibration curves were constructed and the corresponding regression equations were derived. Analysis of the studied drugs in human plasma Volumes from human plasma samples (1 mL) were transferred into 10 mL screw capped tubes then treated with increasing concentrations of CIS and PRP standard solutions (0.1–0.5 μg/mL for CIS and 1.0–5.0 μg/mL for PRP) and mixed well. Each tube reached a volume of 5 mL by completing the volume with methanol. The mixtures were undergone vortex mixing for 10 seconds and centrifugation at 3,500 rpm for half an hour. The clear solution was separated carefully and the supernatants were filtered by syringe filters. About 1 mL volumes from the filtrates was quantitatively transferred into a series of 10 mL volumetric flasks and analyzed as described before in parallel with blank experiment. The content of plasma is determined via the corresponding regression equation. Results Optimization of the chromatographic conditions Figure 2 represents the chromatogram for CIS and PRP synthetic mixture. Experimental trials were made to obtain the best separation. The results were as follows: Choice of column Two different columns were investigated, which are: monolithic column 130 A (100 mm and 4.6 mm i.d.,2 μ particle size) Merck, Germany and Promosil ODS 100 A column (250 × 4.6 mm i.d., 5 μm particle size) Agela Technologies, USA. The monolithic column was preferred as it resulted in well-defined symmetrical peaks, short analysis time, and good resolution (5 min). Promosil ODS column, on the other hand, gave longer retention times (14 min). Appropriate wavelength detection The wavelengths of maximum fluorescence intensity of the studied drugs were found to be at 324 nm as emission wavelength after excitation 230 nm. The values were chosen based on the fluorescence spectra of the two compounds and the relative fluorescence intensity of each compound. Mobile phase composition Trials were made to obtain the optimum mobile phase composition for maximum selectivity and resolution of the developed system. Regarding pH; increasing the value of pH was found to increase the retention time of both drugs. The optimum pH was found to be 4.5 in regards to resolution and number of theoretical plates (Table I). Table I. Chromatographic Conditions Optimization for Separation of a Mixture of CIS and PRP by the Proposed HPLC Method Parameter No. of theoretical plates (N) Resolution (Rs) Tailing factor (T) Selectivity factor (α) CIS PRP CIS PRP Mobile phase pH 4.0 576 2,280 5.413 1.412 1.223 2.0 4.5 576 2,280 5.413 1.412 1.223 2.5 5.0 643 2,305 9.723 1.784 1.202 3.0 6.0 863 1,286 5.449 1.418 1.299 2.0 7.0 930 1,156 5.542 1.458 1.322 4.0 Conc. of phosphate buffer (M) 0.05 533 2,090 7.458 1.422 1.102 1.0 0.1 576 2,280 5.413 1.412 1.223 2.5 0.15 656 2,212 9.806 1.714 1.220 3.0 0.2 594 1,898 6.042 1.384 1.147 2.0 % of methanol (%v/;v) 60%a 510 2,990 18.018 1.423 0.959 3.5 70% 802 2,409 13.587 1.203 1.015 3.0 80% 576 2,280 5.413 1.412 1.223 2.0 85% 480 2,010 4.819 1.211 1.012 2.0 Organic modifier type (60%, v/v) Propanol 500 2,750 0.800 1.400 1.020 4.0 Acetonitrile 490 2,300 1.200 1.300 1.300 3.0 Methanol 576 2,280 5.413 1.412 1.223 2.0 Flow rate (mL/min) 0.8 532 2,140 10.542 1.800 1.200 1.0 1.0 576 2,280 5.413 1.412 1.223 2.0 1.2 552 2,275 1.200 1.300 1.200 3.0 Parameter No. of theoretical plates (N) Resolution (Rs) Tailing factor (T) Selectivity factor (α) CIS PRP CIS PRP Mobile phase pH 4.0 576 2,280 5.413 1.412 1.223 2.0 4.5 576 2,280 5.413 1.412 1.223 2.5 5.0 643 2,305 9.723 1.784 1.202 3.0 6.0 863 1,286 5.449 1.418 1.299 2.0 7.0 930 1,156 5.542 1.458 1.322 4.0 Conc. of phosphate buffer (M) 0.05 533 2,090 7.458 1.422 1.102 1.0 0.1 576 2,280 5.413 1.412 1.223 2.5 0.15 656 2,212 9.806 1.714 1.220 3.0 0.2 594 1,898 6.042 1.384 1.147 2.0 % of methanol (%v/;v) 60%a 510 2,990 18.018 1.423 0.959 3.5 70% 802 2,409 13.587 1.203 1.015 3.0 80% 576 2,280 5.413 1.412 1.223 2.0 85% 480 2,010 4.819 1.211 1.012 2.0 Organic modifier type (60%, v/v) Propanol 500 2,750 0.800 1.400 1.020 4.0 Acetonitrile 490 2,300 1.200 1.300 1.300 3.0 Methanol 576 2,280 5.413 1.412 1.223 2.0 Flow rate (mL/min) 0.8 532 2,140 10.542 1.800 1.200 1.0 1.0 576 2,280 5.413 1.412 1.223 2.0 1.2 552 2,275 1.200 1.300 1.200 3.0 The shaded values show the optimum chromatographic conditions. aLong retention time (11 min). Numberoftheoreticalplates(N)=5.54(tRWh/2)2. Resolution(Rs)=2ΔtRW1+W2. Tailingfactor(T)=W0.052f. Selectivityfactor(relativeretention)(α)=tR2−tmtR1−tm. Table I. Chromatographic Conditions Optimization for Separation of a Mixture of CIS and PRP by the Proposed HPLC Method Parameter No. of theoretical plates (N) Resolution (Rs) Tailing factor (T) Selectivity factor (α) CIS PRP CIS PRP Mobile phase pH 4.0 576 2,280 5.413 1.412 1.223 2.0 4.5 576 2,280 5.413 1.412 1.223 2.5 5.0 643 2,305 9.723 1.784 1.202 3.0 6.0 863 1,286 5.449 1.418 1.299 2.0 7.0 930 1,156 5.542 1.458 1.322 4.0 Conc. of phosphate buffer (M) 0.05 533 2,090 7.458 1.422 1.102 1.0 0.1 576 2,280 5.413 1.412 1.223 2.5 0.15 656 2,212 9.806 1.714 1.220 3.0 0.2 594 1,898 6.042 1.384 1.147 2.0 % of methanol (%v/;v) 60%a 510 2,990 18.018 1.423 0.959 3.5 70% 802 2,409 13.587 1.203 1.015 3.0 80% 576 2,280 5.413 1.412 1.223 2.0 85% 480 2,010 4.819 1.211 1.012 2.0 Organic modifier type (60%, v/v) Propanol 500 2,750 0.800 1.400 1.020 4.0 Acetonitrile 490 2,300 1.200 1.300 1.300 3.0 Methanol 576 2,280 5.413 1.412 1.223 2.0 Flow rate (mL/min) 0.8 532 2,140 10.542 1.800 1.200 1.0 1.0 576 2,280 5.413 1.412 1.223 2.0 1.2 552 2,275 1.200 1.300 1.200 3.0 Parameter No. of theoretical plates (N) Resolution (Rs) Tailing factor (T) Selectivity factor (α) CIS PRP CIS PRP Mobile phase pH 4.0 576 2,280 5.413 1.412 1.223 2.0 4.5 576 2,280 5.413 1.412 1.223 2.5 5.0 643 2,305 9.723 1.784 1.202 3.0 6.0 863 1,286 5.449 1.418 1.299 2.0 7.0 930 1,156 5.542 1.458 1.322 4.0 Conc. of phosphate buffer (M) 0.05 533 2,090 7.458 1.422 1.102 1.0 0.1 576 2,280 5.413 1.412 1.223 2.5 0.15 656 2,212 9.806 1.714 1.220 3.0 0.2 594 1,898 6.042 1.384 1.147 2.0 % of methanol (%v/;v) 60%a 510 2,990 18.018 1.423 0.959 3.5 70% 802 2,409 13.587 1.203 1.015 3.0 80% 576 2,280 5.413 1.412 1.223 2.0 85% 480 2,010 4.819 1.211 1.012 2.0 Organic modifier type (60%, v/v) Propanol 500 2,750 0.800 1.400 1.020 4.0 Acetonitrile 490 2,300 1.200 1.300 1.300 3.0 Methanol 576 2,280 5.413 1.412 1.223 2.0 Flow rate (mL/min) 0.8 532 2,140 10.542 1.800 1.200 1.0 1.0 576 2,280 5.413 1.412 1.223 2.0 1.2 552 2,275 1.200 1.300 1.200 3.0 The shaded values show the optimum chromatographic conditions. aLong retention time (11 min). Numberoftheoreticalplates(N)=5.54(tRWh/2)2. Resolution(Rs)=2ΔtRW1+W2. Tailingfactor(T)=W0.052f. Selectivityfactor(relativeretention)(α)=tR2−tmtR1−tm. The ionic strength of phosphate buffer was considered. It was found that using 0.1 M phosphate buffer resulted in resolved peaks. However, replacement with water resulted in broadening of the two peaks (Table I). Type and ratio of the organic modifier were also studied. The ideal one was methanol among the different studied solvents such as propanol and acetonitrile. Both acetonitrile and propanol caused overlapping of the two peaks. According to the study, decreasing the ratio of methanol caused significant increase in the retention time of PRP. The results are abridged in Table I. Flow rate Optimal separation was achieved upon eluting the mobile phase in the rate of 1.0 mL /min. Increasing the flow rate resulted in decreased resolution of the two peak, and decreasing it resulted in longer retention times (Table I). Validation of the method Linearity A linear ascending relationship was reached by plotting the peak area of each drug versus its concentration (μg/mL) and a linear regression analysis was conducted to illustrate the linearity of the method. The graphs were linear over the specified ranges in Table II. The high values of correlation coefficient in the following equations proved the linearity: P=4.4×105+6.06×106C(r=0.9999)forCISP=−1.4×104+1×106C(r=0.9999)forPRP Table II. Performance Data for the Determination of the CIS and PRP by the Proposed Method Parameter CIS PRP Wavelength [λex. / λem.] (nm) 230 / 324 Linearity range (μg/mL) 0.01–1.0 0.1–3.0 Intercept (a) 4.4 × 105 −1.4 × 104 Slope (b) 6.06 × 106 1 × 106 Correlation coefficient (r) 0.9999 0.9999 S.D. of residuals (Sy/x) 4.91 × 103 1.65 × 104 S.D. of intercept (Sa) 3.52 × 103 9.96 × 103 S.D. of slope (Sb) 5.95 × 103 6.45 × 103 S.D. 0.49 1.39 % RSDa 0.49 1.39 LOD (μg/mL)b 0.002 0.03 LOQ (μg/mL)c 0.006 0.100 Parameter CIS PRP Wavelength [λex. / λem.] (nm) 230 / 324 Linearity range (μg/mL) 0.01–1.0 0.1–3.0 Intercept (a) 4.4 × 105 −1.4 × 104 Slope (b) 6.06 × 106 1 × 106 Correlation coefficient (r) 0.9999 0.9999 S.D. of residuals (Sy/x) 4.91 × 103 1.65 × 104 S.D. of intercept (Sa) 3.52 × 103 9.96 × 103 S.D. of slope (Sb) 5.95 × 103 6.45 × 103 S.D. 0.49 1.39 % RSDa 0.49 1.39 LOD (μg/mL)b 0.002 0.03 LOQ (μg/mL)c 0.006 0.100 aPercentage relative standard deviation. bLimit of detection. cLimit of quantitation. View Large Table II. Performance Data for the Determination of the CIS and PRP by the Proposed Method Parameter CIS PRP Wavelength [λex. / λem.] (nm) 230 / 324 Linearity range (μg/mL) 0.01–1.0 0.1–3.0 Intercept (a) 4.4 × 105 −1.4 × 104 Slope (b) 6.06 × 106 1 × 106 Correlation coefficient (r) 0.9999 0.9999 S.D. of residuals (Sy/x) 4.91 × 103 1.65 × 104 S.D. of intercept (Sa) 3.52 × 103 9.96 × 103 S.D. of slope (Sb) 5.95 × 103 6.45 × 103 S.D. 0.49 1.39 % RSDa 0.49 1.39 LOD (μg/mL)b 0.002 0.03 LOQ (μg/mL)c 0.006 0.100 Parameter CIS PRP Wavelength [λex. / λem.] (nm) 230 / 324 Linearity range (μg/mL) 0.01–1.0 0.1–3.0 Intercept (a) 4.4 × 105 −1.4 × 104 Slope (b) 6.06 × 106 1 × 106 Correlation coefficient (r) 0.9999 0.9999 S.D. of residuals (Sy/x) 4.91 × 103 1.65 × 104 S.D. of intercept (Sa) 3.52 × 103 9.96 × 103 S.D. of slope (Sb) 5.95 × 103 6.45 × 103 S.D. 0.49 1.39 % RSDa 0.49 1.39 LOD (μg/mL)b 0.002 0.03 LOQ (μg/mL)c 0.006 0.100 aPercentage relative standard deviation. bLimit of detection. cLimit of quantitation. View Large P is the peak area, C is the concentration of the drug in μg/mL and r is the correlation coefficient. Statistical analysis (31) of the data resulted in small values of the standard deviation of residuals (Sy/x), of intercept (Sa), and of slope (Sb), and small value of the percentage relative standard deviation and the percentage relative error (Table III). These data pointed out to the linearity of the calibration graph. Table III. Assay Results for the Determination of the Studied Drugs in Pure form by the Proposed and Comparison Methods Compound Proposed method Comparison method (4) Amount taken (μg/mL) Amount found (μg/mL) % Found Amount taken (μg/mL) Amount found (μg/mL) % Found CIS 0.01 0.01 100.0 40.0 39.866 99.67 0.1 0.099 99.0 50.0 50.267 100.53 0.5 0.501 100.20 60.0 59.866 99.78 0.7 0.701 100.14 1.0 0.999 99.90 Mean 99.85 99.99 ±S.D. 0.49 0.35 t 0.312 (2.45) F 1.96 (19.25) PRP 0.1 0.099 99.0 40.0 40.490 101.23 0.2 0.201 100.50 50.0 49.019 98.04 0.5 0.508 101.60 60.0 60.490 100.82 1.0 0.979 97.90 2.0 2.023 101.15 3.0 2.990 99.67 Mean 99.97 100.03 ±S.D. 1.39 1.00 t 0.091 (2.20) F 1.93 (19.25) Compound Proposed method Comparison method (4) Amount taken (μg/mL) Amount found (μg/mL) % Found Amount taken (μg/mL) Amount found (μg/mL) % Found CIS 0.01 0.01 100.0 40.0 39.866 99.67 0.1 0.099 99.0 50.0 50.267 100.53 0.5 0.501 100.20 60.0 59.866 99.78 0.7 0.701 100.14 1.0 0.999 99.90 Mean 99.85 99.99 ±S.D. 0.49 0.35 t 0.312 (2.45) F 1.96 (19.25) PRP 0.1 0.099 99.0 40.0 40.490 101.23 0.2 0.201 100.50 50.0 49.019 98.04 0.5 0.508 101.60 60.0 60.490 100.82 1.0 0.979 97.90 2.0 2.023 101.15 3.0 2.990 99.67 Mean 99.97 100.03 ±S.D. 1.39 1.00 t 0.091 (2.20) F 1.93 (19.25) The values between parenthesis are tabulated t and F values, respectively at P = 0.05 (31). Each result is the average of three separate determinations. Table III. Assay Results for the Determination of the Studied Drugs in Pure form by the Proposed and Comparison Methods Compound Proposed method Comparison method (4) Amount taken (μg/mL) Amount found (μg/mL) % Found Amount taken (μg/mL) Amount found (μg/mL) % Found CIS 0.01 0.01 100.0 40.0 39.866 99.67 0.1 0.099 99.0 50.0 50.267 100.53 0.5 0.501 100.20 60.0 59.866 99.78 0.7 0.701 100.14 1.0 0.999 99.90 Mean 99.85 99.99 ±S.D. 0.49 0.35 t 0.312 (2.45) F 1.96 (19.25) PRP 0.1 0.099 99.0 40.0 40.490 101.23 0.2 0.201 100.50 50.0 49.019 98.04 0.5 0.508 101.60 60.0 60.490 100.82 1.0 0.979 97.90 2.0 2.023 101.15 3.0 2.990 99.67 Mean 99.97 100.03 ±S.D. 1.39 1.00 t 0.091 (2.20) F 1.93 (19.25) Compound Proposed method Comparison method (4) Amount taken (μg/mL) Amount found (μg/mL) % Found Amount taken (μg/mL) Amount found (μg/mL) % Found CIS 0.01 0.01 100.0 40.0 39.866 99.67 0.1 0.099 99.0 50.0 50.267 100.53 0.5 0.501 100.20 60.0 59.866 99.78 0.7 0.701 100.14 1.0 0.999 99.90 Mean 99.85 99.99 ±S.D. 0.49 0.35 t 0.312 (2.45) F 1.96 (19.25) PRP 0.1 0.099 99.0 40.0 40.490 101.23 0.2 0.201 100.50 50.0 49.019 98.04 0.5 0.508 101.60 60.0 60.490 100.82 1.0 0.979 97.90 2.0 2.023 101.15 3.0 2.990 99.67 Mean 99.97 100.03 ±S.D. 1.39 1.00 t 0.091 (2.20) F 1.93 (19.25) The values between parenthesis are tabulated t and F values, respectively at P = 0.05 (31). Each result is the average of three separate determinations. Limit of quantitation and limit of detection Limit of quantitation (LOQ) and limit of detection (LOD) were determined according to ICH guidelines (32) and the results obtained are abridged in Table II. Accuracy To study the suitability and reliability of the method; an HPLC method previously mentioned for CIS and PRP determination (4) was adopted. The comparison method (4) described an HPLC method using a Spherisorb ODS-2 column and a mobile phase consisted of acetonitrile: ammonium formate (50:50) v/v., at 280 nm. The results abridged in Table III point out to the agreement of the results obtained by both methods regarding accuracy as revealed by the student t-test (31). Precision The concentrations used for evaluating the precision were; 0.1, 0.5 and 0.7 μg/mL for CIS, and 0.5, 1.0 and 2.0 μg/mL for PRP. The results were abridged in Table III. Robustness of the HPLC method Some minor variations did not affect the separation of CIS and PRP proving the reliability of the method. These changes were pH (4.5 ± 0.5) and phosphate buffer concentration (0.1 ± 0.005 M). The ratio of methanol and 0.1 M phosphate buffer in the mobile phase were critical, as small variations in such ratio (80:20, v/v) resulted in alteration of the resolution values of both drugs (Table IV). Table IV. Precision Data for the Determination of the CIS and PRP by the Proposed Method Intra-day Inter-day CIS (μg/mL) mean ± S.D % RSD mean ± S.D % RSD 0.1 99.09 ± 0.84 0.84 99.42 ± 0.50 0.50 0.5 98.53 ± 1.40 1.43 98.83 ± 1.12 1.13 0.7 98.37 ± 1.56 1.58 98.42 ± 1.49 1.50 PRP 0.5 98.57 ± 1.76 1.79 98.92 ± 0.95 0.96 1.0 98.82 ± 1.12 1.13 98.76 ± 1.2 1.22 2.0 98.90 ± 1.15 1.17 99.15 ± 0.73 0.74 Intra-day Inter-day CIS (μg/mL) mean ± S.D % RSD mean ± S.D % RSD 0.1 99.09 ± 0.84 0.84 99.42 ± 0.50 0.50 0.5 98.53 ± 1.40 1.43 98.83 ± 1.12 1.13 0.7 98.37 ± 1.56 1.58 98.42 ± 1.49 1.50 PRP 0.5 98.57 ± 1.76 1.79 98.92 ± 0.95 0.96 1.0 98.82 ± 1.12 1.13 98.76 ± 1.2 1.22 2.0 98.90 ± 1.15 1.17 99.15 ± 0.73 0.74 Each result is the average of three separate determinations. Table IV. Precision Data for the Determination of the CIS and PRP by the Proposed Method Intra-day Inter-day CIS (μg/mL) mean ± S.D % RSD mean ± S.D % RSD 0.1 99.09 ± 0.84 0.84 99.42 ± 0.50 0.50 0.5 98.53 ± 1.40 1.43 98.83 ± 1.12 1.13 0.7 98.37 ± 1.56 1.58 98.42 ± 1.49 1.50 PRP 0.5 98.57 ± 1.76 1.79 98.92 ± 0.95 0.96 1.0 98.82 ± 1.12 1.13 98.76 ± 1.2 1.22 2.0 98.90 ± 1.15 1.17 99.15 ± 0.73 0.74 Intra-day Inter-day CIS (μg/mL) mean ± S.D % RSD mean ± S.D % RSD 0.1 99.09 ± 0.84 0.84 99.42 ± 0.50 0.50 0.5 98.53 ± 1.40 1.43 98.83 ± 1.12 1.13 0.7 98.37 ± 1.56 1.58 98.42 ± 1.49 1.50 PRP 0.5 98.57 ± 1.76 1.79 98.92 ± 0.95 0.96 1.0 98.82 ± 1.12 1.13 98.76 ± 1.2 1.22 2.0 98.90 ± 1.15 1.17 99.15 ± 0.73 0.74 Each result is the average of three separate determinations. Pharmaceutical preparations The studied drugs were assayed in their ampoules and the results were satisfactory as in Table V. The results of their assay were in good agreement with those of the comparison method (4). Table V. Assay Results for the Determination of the Studied Drugs in their Different Dosage Forms by Proposed and Comparison Methods Proposed method Comparison method (4) Dosage form Amount taken Amount found % recovery Amount taken Amount found % recovery 1-CIS ampoules (CIS 25 mg /2.5 mL) 0.1 0.102 102.0 40.0 40.134 100.34 0.5 0.494 98.80 50.0 49.731 99.46 0.7 0.704 100.57 60.0 60.134 100.22 Mean 100.46 100.01 ±S.D 1.10 0.36 t 1.000 F 9.336 2-Propofol ampoules (PRP 10 mg/mL) 0.5 0.502 100.4 40.0 40.279 100.70 1.0 0.997 99.7 50.0 49.441 98.88 2.0 2.001 100.05 60.0 60.279 100.47 Mean 100.05 100.02 ±S.D 0.23 0.75 t 0.146 F 10.633 Proposed method Comparison method (4) Dosage form Amount taken Amount found % recovery Amount taken Amount found % recovery 1-CIS ampoules (CIS 25 mg /2.5 mL) 0.1 0.102 102.0 40.0 40.134 100.34 0.5 0.494 98.80 50.0 49.731 99.46 0.7 0.704 100.57 60.0 60.134 100.22 Mean 100.46 100.01 ±S.D 1.10 0.36 t 1.000 F 9.336 2-Propofol ampoules (PRP 10 mg/mL) 0.5 0.502 100.4 40.0 40.279 100.70 1.0 0.997 99.7 50.0 49.441 98.88 2.0 2.001 100.05 60.0 60.279 100.47 Mean 100.05 100.02 ±S.D 0.23 0.75 t 0.146 F 10.633 The value of tabulated t and F are 2.78 and 19.00, respectively at P = 0.05 (31). Each result is the average of three separate determinations. Table V. Assay Results for the Determination of the Studied Drugs in their Different Dosage Forms by Proposed and Comparison Methods Proposed method Comparison method (4) Dosage form Amount taken Amount found % recovery Amount taken Amount found % recovery 1-CIS ampoules (CIS 25 mg /2.5 mL) 0.1 0.102 102.0 40.0 40.134 100.34 0.5 0.494 98.80 50.0 49.731 99.46 0.7 0.704 100.57 60.0 60.134 100.22 Mean 100.46 100.01 ±S.D 1.10 0.36 t 1.000 F 9.336 2-Propofol ampoules (PRP 10 mg/mL) 0.5 0.502 100.4 40.0 40.279 100.70 1.0 0.997 99.7 50.0 49.441 98.88 2.0 2.001 100.05 60.0 60.279 100.47 Mean 100.05 100.02 ±S.D 0.23 0.75 t 0.146 F 10.633 Proposed method Comparison method (4) Dosage form Amount taken Amount found % recovery Amount taken Amount found % recovery 1-CIS ampoules (CIS 25 mg /2.5 mL) 0.1 0.102 102.0 40.0 40.134 100.34 0.5 0.494 98.80 50.0 49.731 99.46 0.7 0.704 100.57 60.0 60.134 100.22 Mean 100.46 100.01 ±S.D 1.10 0.36 t 1.000 F 9.336 2-Propofol ampoules (PRP 10 mg/mL) 0.5 0.502 100.4 40.0 40.279 100.70 1.0 0.997 99.7 50.0 49.441 98.88 2.0 2.001 100.05 60.0 60.279 100.47 Mean 100.05 100.02 ±S.D 0.23 0.75 t 0.146 F 10.633 The value of tabulated t and F are 2.78 and 19.00, respectively at P = 0.05 (31). Each result is the average of three separate determinations. Biological fluids Known amounts of the CIS and PRP were added to aliquots of human plasma samples to obtain various final concentrations ranging from 0.01 to 0.05 μg/mL and from 0.1 to 0.5 respectively. These values were previously reported for the two drugs in human plasma (33, 34). The sensitivity of the proposed method is high enough to determine CIS and PRP in spiked human plasma samples. Precipitation with methanol was adopted for the estimation of CIS and PRP in plasma samples. Under the previously described experimental conditions, a linear relationship was established by plotting the peak area against the drug concentration in μg/mL. The following equations represent the linear regression analysis: P=1316083.600+115944360.000C(r=0.9979)forCISP=−14380.200+974449.000C(r=0.9999)forPRP The high values of the correlation coefficients (r) point out to the good linearity of the plasma calibration graphs. The results for the assay in plasma are summarized in Table VI. The proposed method was applied for the determination of CIS and PRP in spiked human plasma over the concentration range of 0.01–0.05 and 0.10–0.50 μg/mL, respectively. Figure 4 shows representative chromatogram for spiked human plasma sample. Table VI. Results for the Determination of the CIS and PRP in Spiked Human Plasma Samples Using the Proposed Method Matrix Amount added (μg/mL) Amount found (μg/mL) % Recovery CIS PRP CIS PRP CIS PRP Spiked human plasma 0.01 0.10 0.0105 0.098 105.00 98.00 0.02 0.20 0.019 0.204 95.00 102.00 0.03 0.40 0.031 0.397 103.33 99.25 0.05 0.50 0.049 0.501 98.00 100.20 X¯ 100.33 99.86 ±SD ±4.64 ±1.69 % RSD 4.63 1.69 Matrix Amount added (μg/mL) Amount found (μg/mL) % Recovery CIS PRP CIS PRP CIS PRP Spiked human plasma 0.01 0.10 0.0105 0.098 105.00 98.00 0.02 0.20 0.019 0.204 95.00 102.00 0.03 0.40 0.031 0.397 103.33 99.25 0.05 0.50 0.049 0.501 98.00 100.20 X¯ 100.33 99.86 ±SD ±4.64 ±1.69 % RSD 4.63 1.69 Table VI. Results for the Determination of the CIS and PRP in Spiked Human Plasma Samples Using the Proposed Method Matrix Amount added (μg/mL) Amount found (μg/mL) % Recovery CIS PRP CIS PRP CIS PRP Spiked human plasma 0.01 0.10 0.0105 0.098 105.00 98.00 0.02 0.20 0.019 0.204 95.00 102.00 0.03 0.40 0.031 0.397 103.33 99.25 0.05 0.50 0.049 0.501 98.00 100.20 X¯ 100.33 99.86 ±SD ±4.64 ±1.69 % RSD 4.63 1.69 Matrix Amount added (μg/mL) Amount found (μg/mL) % Recovery CIS PRP CIS PRP CIS PRP Spiked human plasma 0.01 0.10 0.0105 0.098 105.00 98.00 0.02 0.20 0.019 0.204 95.00 102.00 0.03 0.40 0.031 0.397 103.33 99.25 0.05 0.50 0.049 0.501 98.00 100.20 X¯ 100.33 99.86 ±SD ±4.64 ±1.69 % RSD 4.63 1.69 Figure 4. View largeDownload slide Typical chromatogram of the studied drugs in spiked human plasma under the described chromatographic conditions: (A) blank plasma. (B) Plasma sample spiked with CIS (0.05 μg/mL) and PRP (0.5 μg/mL). Figure 4. View largeDownload slide Typical chromatogram of the studied drugs in spiked human plasma under the described chromatographic conditions: (A) blank plasma. (B) Plasma sample spiked with CIS (0.05 μg/mL) and PRP (0.5 μg/mL). Discussion There is evidence that using CIS and PRP during surgical operations is frequent due to their synergistic action. CIS is neuromuscular blocking agent. PRP is a short-acting anesthetic which give the best recovery for short time operations that are less than 30 min (35). The literature reported an HPLC method using UV detection for the separation of the two drugs (4). The proposed method has several advantages over the reported one: interestingly, using HPLC coupled with fluorescence detection resulted in increased sensitivity. In addition using monolithic column provided short time of analysis (<5 min). Moreover; the suggested procedure was used successfully to determine the CIS and PRP simultaneously in spiked human plasma (in vitro). Conclusion Referring to the importance of two drugs (CIS and PRP) which are co-administered during surgery, new HPLC methodology has been developed for their simultaneous determination in spiked human plasma. The assay procedure involved the use of HPLC coupled with fluorescence detection. The proposed method is highly sensitive, as down to 2.0 and 30 ng/mL of CIS and PRP could be detected respectively. The monolithic column permits the separation to be performed in <5 min. The method also utilized to the estimation of both compounds in their dosage forms. References 1 Sweetman, S. Martindale . ; (The complete drug reference). In Electronic version . The Pharmaceutical Press , London , ( 2009 ). 2 The United States Pharmacopoeia 34 . , The National Formulary 29; the US Pharmacopoeial Convention: Rockville, MD, ( 2011 ). 3 The British Pharmacopoeia . , Her Magesty’s Stationary Office: London, 2015 ; Vol. II. 4 Zhang , H. , Wang , P. , Bartlett , M.G. , Stewart , J.T. ; HPLC determination of cisatracurium besylate and propofol mixtures with LC-MS identification of degradation products ; Journal of Pharmaceutical and Biomedical Analysis , ( 1998 ); 16 ( 7 ): 1241 – 1249 . Google Scholar CrossRef Search ADS PubMed 5 Bryant , B.J. , James , C.D. , Ryan Cook , D. , Croft Harrelson , J.;. ; High performance liquid chromatographic assay for cisatracurium and its metabolites in human urine ; Journal of Liquid Chromatography & Related Technologies , ( 1997 ); 20 ( 13 ): 2041 – 2051 . Google Scholar CrossRef Search ADS 6 Gao , J. , Yang , T. , Ye , M. , Zhang , X. , Tian , G. , Zhen , Q. , et al. . ; High-performance liquid chromatography assay with programmed flow elution for cisatracurium in human plasma: Application to pharmacokinetics in infants and children ; Journal of Chromatography, B: Biomedical Sciences and Applications , ( 2014 ); 955-956 : 58 – 63 . 7 Li , J. , Chen , B. , Yang , W , Zhang , Y. ; Determination of cisatracurium in human plasma by iron-pair HPLC with fluorescence detection ; Chinese Journal of Pharmaceutical Analysis , ( 2011 ); 31 : 713 – 716 . 8 Błazewicz , A. , Fijałek , Z. , Warowna-Grześkiewicz , M. , Jadach , M. ; Determination of atracurium, cisatracurium and mivacurium with their impurities in pharmaceutical preparations by liquid chromatography with charged aerosol detection ; Journal of Chromatography A , ( 2010 ); 1217 ( 8 ): 1266 – 1272 . Google Scholar CrossRef Search ADS PubMed 9 Rut , F. , Miguel , A.B. , Manuel , C.N. , Juan , C.J. , Nez , A.G. ; Spectrofluorimetric determination of cisatracurium and mivacurium in spiked human serum and pharmaceuticals ; Talanta , ( 1999 ); 49 : 881 – 887 . Google Scholar CrossRef Search ADS PubMed 10 Fernandez , T. , Callejon , M. , Jimenez , S. , Bello Lopez , M.A. , Perez , A.G. ; Electrochemical oxidation of cisatracurium on carbon paste electrode and its analytical applications ; Talanta , ( 2001 ); 53 : 1179 – 1185 . Google Scholar CrossRef Search ADS PubMed 11 Ming , Z. , Jieying , G. , Xiaoqing , Z. , Yue , C. , Zimian , F. , Min , D. ; Capillary electrophoresis with electrochemiluminescence detection for the simultaneous determination of cisatracurium besylate and its degradation products in pharmaceutical preprations: electrodriven separations ; Journal of Separation Science , ( 2015 ); 38 ( 13 ): 2332 – 2339 . Google Scholar CrossRef Search ADS PubMed 12 Pissinis , D. , Sereno , L.E. , Marioli , J.M. ; Multi-wavelength spectrophotometric determination of propofol acidity constant in different acetonitrile-water mixture ; Journal of Brazilian Chemical Society , ( 2005 ); 16 ( 5 ): 1054 – 1060 . Google Scholar CrossRef Search ADS 13 VanaŠ , R. , Célia , G. , Amorim , H.S. , Maria , C.B.M. , Burkhard , H. , Alberto , N.A. , et al. . ; Fully automated analytical procedure for propofol determination by sequential injection technique with spectrophotometric and fluorimetric detections ; Talanta , ( 2014 ); 118 : 104 – 110 . Google Scholar CrossRef Search ADS PubMed 14 Dawidowicz , A.L. , Fijalkowska , A. ; Determination of propofol in blood by HPLC. Comparison of the extraction and precipitation methods ; Journal of Chromatographic Science , ( 1995 ); 33 ( 7 ): 372 – 382 . Google Scholar CrossRef Search ADS 15 King , D.T. , Stewart , J.T. , Venkateshwaran , T.G. ; HPLC determination of propofol-thiopental sodium and propofol-ondansetron mixtures ; Journal of Liquid Chromatography and Related Technology , ( 1996 ); 19 ( 14 ): 2285 – 2294 . Google Scholar CrossRef Search ADS 16 Vishwanathan , K. , Stewart , J.T. ; HPLC determination of a propofol and remifentanil mixture ; Journal of Liquid Chromatography and Related Technology , ( 1999 ); 22 ( 6 ): 923 – 931 . Google Scholar CrossRef Search ADS 17 Yeganeh , M.H. , Ramzan , I. ; Determination of propofol in rat whole blood and plasma by high- performance liquid chromatography ; Journal of Chromatography B , ( 1997 ); 691 ( 2 ): 478 – 482 . Google Scholar CrossRef Search ADS 18 Kwak , J.H. , Hye , K.K. , Sanggli , C. , Sangwhau , L. , Jae , S.P. ; Determination of propofol glucouronide from hair sample by using mixed mode anion exchange cartridge and liquid chromatography mass spectrometry ; Journal of Chromatography B , ( 2016 ); 1015-1016 : 209 – 213 . Google Scholar CrossRef Search ADS 19 Fabio , V. , Giovanni , S. , Mathia , F. , Alessia , F. , Francesco , M. , Elisabetla , B. ; LC-MS/MS and GC-MS methods in propofol detection. Evaluation of two analytical procedures ; Forensic Science International , ( 2015 ); 266 : 1 – 6 . 20 Lambert , K.S. , Jorgen , B.H. ; Simultaneous determination of propfol and its glucouronide in whole blood by liquid chromatography electrospray tandem mass spectrometry and the influence of sample storage conditions on the reliability of the test results ; Journal of Pharmaceutical and Biomedical Analysis , ( 2015 ); 109 : 158 – 163 . Google Scholar CrossRef Search ADS PubMed 21 Kim , H.S. , Cheong , J.C. , Lee , J.I. , In , M.K. ; Rapid and sensitive determination of propofol glucouronide in hair by liquid chromatography and tandem mass spectrometry ; Journal of Pharmaceutical and Biomedical Analysis , ( 2013 ); 85 : 33 – 39 . Google Scholar CrossRef Search ADS PubMed 22 Mariska , Y.M.P. , Hiltjo , K. , Ben , G. , Joukje , V.D.N. , Catherijne , A.J.K. , Donald , R.A.U. ; Gas chromatography mass spectrometric assay for propofol in cerebrospinal fluid of traumatic brain patients ; Journal of Chromatography B , ( 2007 ); 852 : 635 – 639 . Google Scholar CrossRef Search ADS 23 Francis , B. , Sarah , A.G. , Andrew , W. , Jean , F.M. , Pascal , V. ; Development of a rapid and sensitive LC-ESI/MS/MS assay for the quantification of propofol using a simple offline dansyl chloride derivatization reaction to enhance signal intensity ; Journal of Pharmaceutical and Biomedical Analysis , ( 2005 ); 39 : 411 – 417 . Google Scholar CrossRef Search ADS PubMed 24 Knibbe , C.A.J. , Koster , V.S. , Deneer , V.H.M. , Stuurman , R.M. , Kuks , P.F.M. , Lange , R. ; Determination of propofol in low-volume samples by high-performance liquid chromatography with fluorescence detection ; Journal of Chromatography B , ( 1998 ); 706 : 305 – 310 . Google Scholar CrossRef Search ADS 25 Tadashi , N. , Rie , S. , Yuko , T. , Hideko , K. , Teruo , O. , Takako , M.N. ; Aqueous chromatographic system for the quantification of propofol in biological fluids using a temperature responsive polymer modified stationary phase ; Journal of Chromatography A , ( 2009 ); 1216 : 7427 – 7432 . Google Scholar CrossRef Search ADS PubMed 26 Sun , Y.L. , Na-Hyun , P. , Eun-Kyung , J. , Jae-Woo , W. , Chang-Ju , K. , MoonKuo , I. , et al. . ; Comparison of GC/MS and LC/MS methods for the analysis of propfol and its metabolites in urine ; Journal of Chromatography B , ( 2012 ); 900 : 1 – 10 . Google Scholar CrossRef Search ADS 27 Si-Cheng , L. , Guang-Bo , G. , Hui-Xin , L. , Hai-Tao , S. , Hong , W. , Zhong-Ze , F. , et al. . ; Determination of propofol UDP-glucuronosyltransferase (UGT) activities in hepatic microsomes from different species by UFLC-ESI-MS ; Journal of Pharmaceutical and Biomedical Analysis , ( 2011 ); 54 : 236 – 241 . Google Scholar CrossRef Search ADS PubMed 28 Cohen , S. , Lhuillier , F. , Mouloua , Y. , Vignal , B. , Favetta , P. , Guitton , J. ; Quantitative measurement of propofol and in main glucuroconjugate metabolites in human plasma using solid phase extraction-liquid chromatography-tandem mass spectrometry ; Journal of Chromatography B , ( 2007 ); 854 : 165 – 172 . Google Scholar CrossRef Search ADS 29 Jan , L. , Fernando , G. , Fracine , K. , Edward , C. , Erno , L. ; Electrochemical quantification of 2,6-diisopropylphenol(propofol) ; Analytica Chimica Acta , ( 2011 ); 704 : 63 – 67 . Google Scholar CrossRef Search ADS PubMed 30 Nathan , N. , Bonada , G. , Feiss , P. ; Potentiation of atracurium by pancuronium during propofol-fentanyl-N2O anesthesia ; Acta Anaesthesiol Belgica , ( 1996 ); 47 ( 4 ): 187 – 193 . 31 Miller , J.N. , Miller , J.C. ; Statistics and Chemometrics for Analytical Chemistry, Harlow , 5th ed. . Pearson Education Limited , Edinburgh Gate, Harlow , ( 2005 ); pp. 39 – 73 . 107-149, 256. 32 ICH Harmonized Tripartite Guidelines . , Validation of Analytical Procedures: Text and Methodology, Q2(R1), Current Step 4 Version, Parent Guidelines on Methodology Dated November 6; 1996, Incorporated in November ( 2005 ). 33 Moffat , A.C. , Osselton , M.D. , Widdop , B. , Galichet , L.Y. ; Clark’s Analysis of Drugs and Poisons in Pharmaceuticals, Body Fluids and Postmortem Material , Vol II , 4th ed. . The Pharmaceutical Press , London , ( 2011 ); pp. 1591 – 1698 . 34 Zhang , T. , Cui , Y. ; Determination of propofol in human plasma by microemulsion liquid chromatography; Chinese ; Journal of Chromatography , ( 2011 ); 29 ( 8 ): 768 – 772 . 35 Aronson , J.K. ; Meyler’s Side Effects of Drugs Used in Anesthesia , 198 . Elsevier Science & Technology , Oxford , ( 2009 ); pp. 63 – 78 . © 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

Simultaneous HPLC Determination of Cisatracurium and Propofol in Human Plasma via Fluorometric Detection

Loading next page...
 
/lp/ou_press/simultaneous-hplc-determination-of-cisatracurium-and-propofol-in-human-3cohZytK6e
Publisher
Oxford University Press
Copyright
© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
ISSN
0021-9665
eISSN
1945-239X
D.O.I.
10.1093/chromsci/bmy027
Publisher site
See Article on Publisher Site

Abstract

Abstract The proposed method describes a high performance liquid chromatographic method with fluoremetric detection for the determination of cisatracurium (CIS) and propofol (PRP) simultaneously, which are co-administered as a pre-operative injection mixture. The separation of the two drugs was achieved using monolithic column (100 mm and 4.6 mm internal diameter) and mixture of methanol and 0.1 M phosphate buffer in the ratio of 80:20 (v/v) at pH 4.5 as a mobile phase. The fluorescence detection was carried out at 230/324 nm. The procedure showed good linearity through the concentration ranges of 0.01–1.00 μg/mL and 0.1–3.0 μg/mL with limits of detection of 0.002, 0.030 μg/mL and limits of quantification of 0.006, 0.100 μg/mL for CIS and PRP, respectively. Simultaneous determination of CIS and PRP in spiked human plasma samples was additionally executed and the results were satisfactory precise and accurate. Introduction Cisatracurium (CIS, Figure 1a) is a competitive neuromuscular blocker that acts by competing with acetylcholine for receptors on the motor end-plate of the neuromuscular junctions to produce blockade. Restoration of normal neuromuscular function can be hastened by increasing the concentration of acetylcholine at the motor end-plate by giving an anticholinesterase such as neostigmine (1). Chemically it is (1 R,1′R,R,2′R)-2,2-[1,5-pentanediylbis-[oxy(3-oxo-3,1-propanediyl)]]-bis[1-[(3,4-dimethoxyphenyl)-methyl]-1,2,3,4-tetrahydro-6,7–dimethoxy-2-methylisoquinolin-ium] dibenzene sulfonate (2). Regarding official methods; the USP monograph (2), and the BP monograph (3), suggested LC method with UV detection at 280 nm for estimation of CIS raw material. The literature revealed limited methods for CIS determination as HPLC (4–8), spectrofluorimetry (9), voltammetry (10) and capillary electrophoresis (11). Figure 1. View largeDownload slide The structural formulae for the studied drugs. Figure 1. View largeDownload slide The structural formulae for the studied drugs. Propofol (PRP, Figure 1b) is 2,6-Bis(1-methylethyl)phenol (2). It acts as a short-acting anesthetic given intravenously to induce and also maintain general anesthesia. It is prescribed for sedation in adult patients undergoing diagnostic procedures, in those undergoing surgery with local or regional anesthesia, and in ventilated adult patients under intensive care. When used for anesthesia, induction is rapid, as is recovery. Propofol has no analgesic effect, so supplementary analgesia may be used (1). The USP recommended gas chromatography equipped with a flame-ionization detector (2) and a LC method was mentioned in the BP for PRP assay (3). Previous publications for the determination of PRP have been reported such as; spectrophotometric analysis (12), sequential injection analysis (13), HPLC (4, 14–28) and electrochemical methods (29). Reviewing the literature indicating; only one research was found in the literature for the analysis of the CIS and PRP using HPLC with UV detection and MS for the identification of the degradation products (4). Combination of CIS and PRP is commonly used during surgery as a pre-operative procedure (30), as CIS besylate is a neuromuscular blocking agent, used as an adjunct to general anesthesia (1). Propofol on the other hand is a general anesthetic, prescribed for induction and maintenance of general anesthesia. Co-administration of the two drugs during surgery is frequent, so our aim was developing a reliable method with reaching low detection limit that permits their determination in human plasma for the purpose of therapeutic drug monitoring. HPLC coupled with fluorescence detection proved to be a reliable technique in such cases. Since the two drugs should be observed together after anesthesia, the method can be adopted for their determination in spiked human plasma. Experimental Equipment HPLC experiments were done using a Shimadzu LC-20AD Chromatograph (Japan) equipped with a Rheodyne injector valve and a 20 μL loop and a RF-10AXL fluorescence detector. A Consort NV P-901 pH –Meter (Belgium) was used for pH measurements. Materials and reagents - CIS with a purity of 99.2% was provided by National Organization for Drug Control and Research (NODCAR), Cairo, Egypt. (The purity was specified by the organization) - Propofol with a purity of 99.90% (as stated by the manufacture) was also kindly provided by NODCAR, Cairo, Egypt. - CIS ampoules, batch # 15023, labeled to contain 25.0 mg /2.5 mL, (product of Hameln Pharmaceutical Germany, for Sunny Medical Group, Cairo, Egypt). - Propofol ampoules, batch # 250127, labeled to contain 10 mg/1 ml, (product of Astrazeneca Pharmaceutical Company, Cairo, Egypt). - Methanol, acetonitrile and n-propanol (HPLC grade) were purchased from Sigma-Aldrich (Germany). - Sodium hydroxide and sodium dihydrogen phosphate were purchased from ADWIC. (Cairo, Egypt). - Orthophosphoric acid (85%, w/v) was purchased from Riedel-deHäen (Sleeze, Germany). - Plasma samples were obtained from Blood-Bank, Mansoura University Hospital, Mansoura, Egypt and were kept frozen until use after gentle thawing. Chromatographic conditions The column used for separation was Chromolith Reversed Phase Monolithic column (100 mm × 4.6 mm i.d.), Merck, Germany. Mobile phase used containing methanol and 0.1 M orthophosphoric acid buffer in the ratio of 80:20, v/v respectively at pH 4.5 was used. Detection wavelengths were 230 nm (excitation) and 324 nm (emission). The separation was done using flow rate 1 mL/min. Standard solutions Stock solutions were prepared by dissolving 1.0 mg/mL of either CIS or PRP in methanol, and completing the volume to 100 mL in a volumetric flask. General recommended procedures Calibration graphs construction Aliquots were accurately transferred to 10 mL volumetric flasks, completed to volume with the mobile phase. The final concentrations would be in the range of 0.01–1.0 μg/mL for CIS and 0.1-3.0 μg/mL for PRP. The average peak areas were plotted against the final concentration of the drug in μg/mL to get the calibration graphs. Then, the regression equations were derived. Analysis of CIS/PRP synthetic mixtures Mixtures of variable ratios of CIS and PRP working standard solutions were transferred into a set of 10 mL volumetric flasks. The procedure described under “Construction of calibration graphs” was carried out. By referring to the calibration graphs; the percentage recovery for each concentration of the drugs was calculated. Analysis of the studied drugs in their ampoules Analysis of CIS in ampoules Five CIS ampoules were emptied and mixed. An accurately measured volume equivalent to 25.0 mg of CIS was transferred into a clean dry 100 mL volumetric flask. The flask was completed with methanol to the mark. Dilution with methanol was done to obtain working solutions within the studied concentration range. The general procedure described under “Construction of Calibration Graphs” was performed. The content of ampoules was then calculated referring to the regression equation. Analysis of PRP in ampoules A volume from PRP ampoules containing 10.0 mg of PRP was transferred into 100 mL volumetric flask, and then the same steps for CIS ampoules were followed (Figure 2). Figure 2. View largeDownload slide Typical chromatogram of synthetic mixture under the described chromatographic conditions: (A): CIS (0.1 μg/mL). (B): PRP (0.5 μg/mL). Figure 2. View largeDownload slide Typical chromatogram of synthetic mixture under the described chromatographic conditions: (A): CIS (0.1 μg/mL). (B): PRP (0.5 μg/mL). Figure 3 shows the chromatogram for CIS and PRP ampoules using the described method. Figure 3. View largeDownload slide Typical chromatogram of ampoules of the studied drugs under the described chromatographic conditions: (A): CIS (0.1 μg/mL). (B): PRP (0.5 μg/mL). Figure 3. View largeDownload slide Typical chromatogram of ampoules of the studied drugs under the described chromatographic conditions: (A): CIS (0.1 μg/mL). (B): PRP (0.5 μg/mL). Analysis of drugs in human plasma Construction of calibration graphs in spiked human plasma Standard calibration graphs were constructed for each of CIS and PRP in human plasma in the ranges of (0.01–0.05 μg/mL for CIS and 0.1–0.5 μg/mL for PRP) by adding appropriate volumes of CIS and PRP working solutions into plasma samples. The calibration curves were constructed and the corresponding regression equations were derived. Analysis of the studied drugs in human plasma Volumes from human plasma samples (1 mL) were transferred into 10 mL screw capped tubes then treated with increasing concentrations of CIS and PRP standard solutions (0.1–0.5 μg/mL for CIS and 1.0–5.0 μg/mL for PRP) and mixed well. Each tube reached a volume of 5 mL by completing the volume with methanol. The mixtures were undergone vortex mixing for 10 seconds and centrifugation at 3,500 rpm for half an hour. The clear solution was separated carefully and the supernatants were filtered by syringe filters. About 1 mL volumes from the filtrates was quantitatively transferred into a series of 10 mL volumetric flasks and analyzed as described before in parallel with blank experiment. The content of plasma is determined via the corresponding regression equation. Results Optimization of the chromatographic conditions Figure 2 represents the chromatogram for CIS and PRP synthetic mixture. Experimental trials were made to obtain the best separation. The results were as follows: Choice of column Two different columns were investigated, which are: monolithic column 130 A (100 mm and 4.6 mm i.d.,2 μ particle size) Merck, Germany and Promosil ODS 100 A column (250 × 4.6 mm i.d., 5 μm particle size) Agela Technologies, USA. The monolithic column was preferred as it resulted in well-defined symmetrical peaks, short analysis time, and good resolution (5 min). Promosil ODS column, on the other hand, gave longer retention times (14 min). Appropriate wavelength detection The wavelengths of maximum fluorescence intensity of the studied drugs were found to be at 324 nm as emission wavelength after excitation 230 nm. The values were chosen based on the fluorescence spectra of the two compounds and the relative fluorescence intensity of each compound. Mobile phase composition Trials were made to obtain the optimum mobile phase composition for maximum selectivity and resolution of the developed system. Regarding pH; increasing the value of pH was found to increase the retention time of both drugs. The optimum pH was found to be 4.5 in regards to resolution and number of theoretical plates (Table I). Table I. Chromatographic Conditions Optimization for Separation of a Mixture of CIS and PRP by the Proposed HPLC Method Parameter No. of theoretical plates (N) Resolution (Rs) Tailing factor (T) Selectivity factor (α) CIS PRP CIS PRP Mobile phase pH 4.0 576 2,280 5.413 1.412 1.223 2.0 4.5 576 2,280 5.413 1.412 1.223 2.5 5.0 643 2,305 9.723 1.784 1.202 3.0 6.0 863 1,286 5.449 1.418 1.299 2.0 7.0 930 1,156 5.542 1.458 1.322 4.0 Conc. of phosphate buffer (M) 0.05 533 2,090 7.458 1.422 1.102 1.0 0.1 576 2,280 5.413 1.412 1.223 2.5 0.15 656 2,212 9.806 1.714 1.220 3.0 0.2 594 1,898 6.042 1.384 1.147 2.0 % of methanol (%v/;v) 60%a 510 2,990 18.018 1.423 0.959 3.5 70% 802 2,409 13.587 1.203 1.015 3.0 80% 576 2,280 5.413 1.412 1.223 2.0 85% 480 2,010 4.819 1.211 1.012 2.0 Organic modifier type (60%, v/v) Propanol 500 2,750 0.800 1.400 1.020 4.0 Acetonitrile 490 2,300 1.200 1.300 1.300 3.0 Methanol 576 2,280 5.413 1.412 1.223 2.0 Flow rate (mL/min) 0.8 532 2,140 10.542 1.800 1.200 1.0 1.0 576 2,280 5.413 1.412 1.223 2.0 1.2 552 2,275 1.200 1.300 1.200 3.0 Parameter No. of theoretical plates (N) Resolution (Rs) Tailing factor (T) Selectivity factor (α) CIS PRP CIS PRP Mobile phase pH 4.0 576 2,280 5.413 1.412 1.223 2.0 4.5 576 2,280 5.413 1.412 1.223 2.5 5.0 643 2,305 9.723 1.784 1.202 3.0 6.0 863 1,286 5.449 1.418 1.299 2.0 7.0 930 1,156 5.542 1.458 1.322 4.0 Conc. of phosphate buffer (M) 0.05 533 2,090 7.458 1.422 1.102 1.0 0.1 576 2,280 5.413 1.412 1.223 2.5 0.15 656 2,212 9.806 1.714 1.220 3.0 0.2 594 1,898 6.042 1.384 1.147 2.0 % of methanol (%v/;v) 60%a 510 2,990 18.018 1.423 0.959 3.5 70% 802 2,409 13.587 1.203 1.015 3.0 80% 576 2,280 5.413 1.412 1.223 2.0 85% 480 2,010 4.819 1.211 1.012 2.0 Organic modifier type (60%, v/v) Propanol 500 2,750 0.800 1.400 1.020 4.0 Acetonitrile 490 2,300 1.200 1.300 1.300 3.0 Methanol 576 2,280 5.413 1.412 1.223 2.0 Flow rate (mL/min) 0.8 532 2,140 10.542 1.800 1.200 1.0 1.0 576 2,280 5.413 1.412 1.223 2.0 1.2 552 2,275 1.200 1.300 1.200 3.0 The shaded values show the optimum chromatographic conditions. aLong retention time (11 min). Numberoftheoreticalplates(N)=5.54(tRWh/2)2. Resolution(Rs)=2ΔtRW1+W2. Tailingfactor(T)=W0.052f. Selectivityfactor(relativeretention)(α)=tR2−tmtR1−tm. Table I. Chromatographic Conditions Optimization for Separation of a Mixture of CIS and PRP by the Proposed HPLC Method Parameter No. of theoretical plates (N) Resolution (Rs) Tailing factor (T) Selectivity factor (α) CIS PRP CIS PRP Mobile phase pH 4.0 576 2,280 5.413 1.412 1.223 2.0 4.5 576 2,280 5.413 1.412 1.223 2.5 5.0 643 2,305 9.723 1.784 1.202 3.0 6.0 863 1,286 5.449 1.418 1.299 2.0 7.0 930 1,156 5.542 1.458 1.322 4.0 Conc. of phosphate buffer (M) 0.05 533 2,090 7.458 1.422 1.102 1.0 0.1 576 2,280 5.413 1.412 1.223 2.5 0.15 656 2,212 9.806 1.714 1.220 3.0 0.2 594 1,898 6.042 1.384 1.147 2.0 % of methanol (%v/;v) 60%a 510 2,990 18.018 1.423 0.959 3.5 70% 802 2,409 13.587 1.203 1.015 3.0 80% 576 2,280 5.413 1.412 1.223 2.0 85% 480 2,010 4.819 1.211 1.012 2.0 Organic modifier type (60%, v/v) Propanol 500 2,750 0.800 1.400 1.020 4.0 Acetonitrile 490 2,300 1.200 1.300 1.300 3.0 Methanol 576 2,280 5.413 1.412 1.223 2.0 Flow rate (mL/min) 0.8 532 2,140 10.542 1.800 1.200 1.0 1.0 576 2,280 5.413 1.412 1.223 2.0 1.2 552 2,275 1.200 1.300 1.200 3.0 Parameter No. of theoretical plates (N) Resolution (Rs) Tailing factor (T) Selectivity factor (α) CIS PRP CIS PRP Mobile phase pH 4.0 576 2,280 5.413 1.412 1.223 2.0 4.5 576 2,280 5.413 1.412 1.223 2.5 5.0 643 2,305 9.723 1.784 1.202 3.0 6.0 863 1,286 5.449 1.418 1.299 2.0 7.0 930 1,156 5.542 1.458 1.322 4.0 Conc. of phosphate buffer (M) 0.05 533 2,090 7.458 1.422 1.102 1.0 0.1 576 2,280 5.413 1.412 1.223 2.5 0.15 656 2,212 9.806 1.714 1.220 3.0 0.2 594 1,898 6.042 1.384 1.147 2.0 % of methanol (%v/;v) 60%a 510 2,990 18.018 1.423 0.959 3.5 70% 802 2,409 13.587 1.203 1.015 3.0 80% 576 2,280 5.413 1.412 1.223 2.0 85% 480 2,010 4.819 1.211 1.012 2.0 Organic modifier type (60%, v/v) Propanol 500 2,750 0.800 1.400 1.020 4.0 Acetonitrile 490 2,300 1.200 1.300 1.300 3.0 Methanol 576 2,280 5.413 1.412 1.223 2.0 Flow rate (mL/min) 0.8 532 2,140 10.542 1.800 1.200 1.0 1.0 576 2,280 5.413 1.412 1.223 2.0 1.2 552 2,275 1.200 1.300 1.200 3.0 The shaded values show the optimum chromatographic conditions. aLong retention time (11 min). Numberoftheoreticalplates(N)=5.54(tRWh/2)2. Resolution(Rs)=2ΔtRW1+W2. Tailingfactor(T)=W0.052f. Selectivityfactor(relativeretention)(α)=tR2−tmtR1−tm. The ionic strength of phosphate buffer was considered. It was found that using 0.1 M phosphate buffer resulted in resolved peaks. However, replacement with water resulted in broadening of the two peaks (Table I). Type and ratio of the organic modifier were also studied. The ideal one was methanol among the different studied solvents such as propanol and acetonitrile. Both acetonitrile and propanol caused overlapping of the two peaks. According to the study, decreasing the ratio of methanol caused significant increase in the retention time of PRP. The results are abridged in Table I. Flow rate Optimal separation was achieved upon eluting the mobile phase in the rate of 1.0 mL /min. Increasing the flow rate resulted in decreased resolution of the two peak, and decreasing it resulted in longer retention times (Table I). Validation of the method Linearity A linear ascending relationship was reached by plotting the peak area of each drug versus its concentration (μg/mL) and a linear regression analysis was conducted to illustrate the linearity of the method. The graphs were linear over the specified ranges in Table II. The high values of correlation coefficient in the following equations proved the linearity: P=4.4×105+6.06×106C(r=0.9999)forCISP=−1.4×104+1×106C(r=0.9999)forPRP Table II. Performance Data for the Determination of the CIS and PRP by the Proposed Method Parameter CIS PRP Wavelength [λex. / λem.] (nm) 230 / 324 Linearity range (μg/mL) 0.01–1.0 0.1–3.0 Intercept (a) 4.4 × 105 −1.4 × 104 Slope (b) 6.06 × 106 1 × 106 Correlation coefficient (r) 0.9999 0.9999 S.D. of residuals (Sy/x) 4.91 × 103 1.65 × 104 S.D. of intercept (Sa) 3.52 × 103 9.96 × 103 S.D. of slope (Sb) 5.95 × 103 6.45 × 103 S.D. 0.49 1.39 % RSDa 0.49 1.39 LOD (μg/mL)b 0.002 0.03 LOQ (μg/mL)c 0.006 0.100 Parameter CIS PRP Wavelength [λex. / λem.] (nm) 230 / 324 Linearity range (μg/mL) 0.01–1.0 0.1–3.0 Intercept (a) 4.4 × 105 −1.4 × 104 Slope (b) 6.06 × 106 1 × 106 Correlation coefficient (r) 0.9999 0.9999 S.D. of residuals (Sy/x) 4.91 × 103 1.65 × 104 S.D. of intercept (Sa) 3.52 × 103 9.96 × 103 S.D. of slope (Sb) 5.95 × 103 6.45 × 103 S.D. 0.49 1.39 % RSDa 0.49 1.39 LOD (μg/mL)b 0.002 0.03 LOQ (μg/mL)c 0.006 0.100 aPercentage relative standard deviation. bLimit of detection. cLimit of quantitation. View Large Table II. Performance Data for the Determination of the CIS and PRP by the Proposed Method Parameter CIS PRP Wavelength [λex. / λem.] (nm) 230 / 324 Linearity range (μg/mL) 0.01–1.0 0.1–3.0 Intercept (a) 4.4 × 105 −1.4 × 104 Slope (b) 6.06 × 106 1 × 106 Correlation coefficient (r) 0.9999 0.9999 S.D. of residuals (Sy/x) 4.91 × 103 1.65 × 104 S.D. of intercept (Sa) 3.52 × 103 9.96 × 103 S.D. of slope (Sb) 5.95 × 103 6.45 × 103 S.D. 0.49 1.39 % RSDa 0.49 1.39 LOD (μg/mL)b 0.002 0.03 LOQ (μg/mL)c 0.006 0.100 Parameter CIS PRP Wavelength [λex. / λem.] (nm) 230 / 324 Linearity range (μg/mL) 0.01–1.0 0.1–3.0 Intercept (a) 4.4 × 105 −1.4 × 104 Slope (b) 6.06 × 106 1 × 106 Correlation coefficient (r) 0.9999 0.9999 S.D. of residuals (Sy/x) 4.91 × 103 1.65 × 104 S.D. of intercept (Sa) 3.52 × 103 9.96 × 103 S.D. of slope (Sb) 5.95 × 103 6.45 × 103 S.D. 0.49 1.39 % RSDa 0.49 1.39 LOD (μg/mL)b 0.002 0.03 LOQ (μg/mL)c 0.006 0.100 aPercentage relative standard deviation. bLimit of detection. cLimit of quantitation. View Large P is the peak area, C is the concentration of the drug in μg/mL and r is the correlation coefficient. Statistical analysis (31) of the data resulted in small values of the standard deviation of residuals (Sy/x), of intercept (Sa), and of slope (Sb), and small value of the percentage relative standard deviation and the percentage relative error (Table III). These data pointed out to the linearity of the calibration graph. Table III. Assay Results for the Determination of the Studied Drugs in Pure form by the Proposed and Comparison Methods Compound Proposed method Comparison method (4) Amount taken (μg/mL) Amount found (μg/mL) % Found Amount taken (μg/mL) Amount found (μg/mL) % Found CIS 0.01 0.01 100.0 40.0 39.866 99.67 0.1 0.099 99.0 50.0 50.267 100.53 0.5 0.501 100.20 60.0 59.866 99.78 0.7 0.701 100.14 1.0 0.999 99.90 Mean 99.85 99.99 ±S.D. 0.49 0.35 t 0.312 (2.45) F 1.96 (19.25) PRP 0.1 0.099 99.0 40.0 40.490 101.23 0.2 0.201 100.50 50.0 49.019 98.04 0.5 0.508 101.60 60.0 60.490 100.82 1.0 0.979 97.90 2.0 2.023 101.15 3.0 2.990 99.67 Mean 99.97 100.03 ±S.D. 1.39 1.00 t 0.091 (2.20) F 1.93 (19.25) Compound Proposed method Comparison method (4) Amount taken (μg/mL) Amount found (μg/mL) % Found Amount taken (μg/mL) Amount found (μg/mL) % Found CIS 0.01 0.01 100.0 40.0 39.866 99.67 0.1 0.099 99.0 50.0 50.267 100.53 0.5 0.501 100.20 60.0 59.866 99.78 0.7 0.701 100.14 1.0 0.999 99.90 Mean 99.85 99.99 ±S.D. 0.49 0.35 t 0.312 (2.45) F 1.96 (19.25) PRP 0.1 0.099 99.0 40.0 40.490 101.23 0.2 0.201 100.50 50.0 49.019 98.04 0.5 0.508 101.60 60.0 60.490 100.82 1.0 0.979 97.90 2.0 2.023 101.15 3.0 2.990 99.67 Mean 99.97 100.03 ±S.D. 1.39 1.00 t 0.091 (2.20) F 1.93 (19.25) The values between parenthesis are tabulated t and F values, respectively at P = 0.05 (31). Each result is the average of three separate determinations. Table III. Assay Results for the Determination of the Studied Drugs in Pure form by the Proposed and Comparison Methods Compound Proposed method Comparison method (4) Amount taken (μg/mL) Amount found (μg/mL) % Found Amount taken (μg/mL) Amount found (μg/mL) % Found CIS 0.01 0.01 100.0 40.0 39.866 99.67 0.1 0.099 99.0 50.0 50.267 100.53 0.5 0.501 100.20 60.0 59.866 99.78 0.7 0.701 100.14 1.0 0.999 99.90 Mean 99.85 99.99 ±S.D. 0.49 0.35 t 0.312 (2.45) F 1.96 (19.25) PRP 0.1 0.099 99.0 40.0 40.490 101.23 0.2 0.201 100.50 50.0 49.019 98.04 0.5 0.508 101.60 60.0 60.490 100.82 1.0 0.979 97.90 2.0 2.023 101.15 3.0 2.990 99.67 Mean 99.97 100.03 ±S.D. 1.39 1.00 t 0.091 (2.20) F 1.93 (19.25) Compound Proposed method Comparison method (4) Amount taken (μg/mL) Amount found (μg/mL) % Found Amount taken (μg/mL) Amount found (μg/mL) % Found CIS 0.01 0.01 100.0 40.0 39.866 99.67 0.1 0.099 99.0 50.0 50.267 100.53 0.5 0.501 100.20 60.0 59.866 99.78 0.7 0.701 100.14 1.0 0.999 99.90 Mean 99.85 99.99 ±S.D. 0.49 0.35 t 0.312 (2.45) F 1.96 (19.25) PRP 0.1 0.099 99.0 40.0 40.490 101.23 0.2 0.201 100.50 50.0 49.019 98.04 0.5 0.508 101.60 60.0 60.490 100.82 1.0 0.979 97.90 2.0 2.023 101.15 3.0 2.990 99.67 Mean 99.97 100.03 ±S.D. 1.39 1.00 t 0.091 (2.20) F 1.93 (19.25) The values between parenthesis are tabulated t and F values, respectively at P = 0.05 (31). Each result is the average of three separate determinations. Limit of quantitation and limit of detection Limit of quantitation (LOQ) and limit of detection (LOD) were determined according to ICH guidelines (32) and the results obtained are abridged in Table II. Accuracy To study the suitability and reliability of the method; an HPLC method previously mentioned for CIS and PRP determination (4) was adopted. The comparison method (4) described an HPLC method using a Spherisorb ODS-2 column and a mobile phase consisted of acetonitrile: ammonium formate (50:50) v/v., at 280 nm. The results abridged in Table III point out to the agreement of the results obtained by both methods regarding accuracy as revealed by the student t-test (31). Precision The concentrations used for evaluating the precision were; 0.1, 0.5 and 0.7 μg/mL for CIS, and 0.5, 1.0 and 2.0 μg/mL for PRP. The results were abridged in Table III. Robustness of the HPLC method Some minor variations did not affect the separation of CIS and PRP proving the reliability of the method. These changes were pH (4.5 ± 0.5) and phosphate buffer concentration (0.1 ± 0.005 M). The ratio of methanol and 0.1 M phosphate buffer in the mobile phase were critical, as small variations in such ratio (80:20, v/v) resulted in alteration of the resolution values of both drugs (Table IV). Table IV. Precision Data for the Determination of the CIS and PRP by the Proposed Method Intra-day Inter-day CIS (μg/mL) mean ± S.D % RSD mean ± S.D % RSD 0.1 99.09 ± 0.84 0.84 99.42 ± 0.50 0.50 0.5 98.53 ± 1.40 1.43 98.83 ± 1.12 1.13 0.7 98.37 ± 1.56 1.58 98.42 ± 1.49 1.50 PRP 0.5 98.57 ± 1.76 1.79 98.92 ± 0.95 0.96 1.0 98.82 ± 1.12 1.13 98.76 ± 1.2 1.22 2.0 98.90 ± 1.15 1.17 99.15 ± 0.73 0.74 Intra-day Inter-day CIS (μg/mL) mean ± S.D % RSD mean ± S.D % RSD 0.1 99.09 ± 0.84 0.84 99.42 ± 0.50 0.50 0.5 98.53 ± 1.40 1.43 98.83 ± 1.12 1.13 0.7 98.37 ± 1.56 1.58 98.42 ± 1.49 1.50 PRP 0.5 98.57 ± 1.76 1.79 98.92 ± 0.95 0.96 1.0 98.82 ± 1.12 1.13 98.76 ± 1.2 1.22 2.0 98.90 ± 1.15 1.17 99.15 ± 0.73 0.74 Each result is the average of three separate determinations. Table IV. Precision Data for the Determination of the CIS and PRP by the Proposed Method Intra-day Inter-day CIS (μg/mL) mean ± S.D % RSD mean ± S.D % RSD 0.1 99.09 ± 0.84 0.84 99.42 ± 0.50 0.50 0.5 98.53 ± 1.40 1.43 98.83 ± 1.12 1.13 0.7 98.37 ± 1.56 1.58 98.42 ± 1.49 1.50 PRP 0.5 98.57 ± 1.76 1.79 98.92 ± 0.95 0.96 1.0 98.82 ± 1.12 1.13 98.76 ± 1.2 1.22 2.0 98.90 ± 1.15 1.17 99.15 ± 0.73 0.74 Intra-day Inter-day CIS (μg/mL) mean ± S.D % RSD mean ± S.D % RSD 0.1 99.09 ± 0.84 0.84 99.42 ± 0.50 0.50 0.5 98.53 ± 1.40 1.43 98.83 ± 1.12 1.13 0.7 98.37 ± 1.56 1.58 98.42 ± 1.49 1.50 PRP 0.5 98.57 ± 1.76 1.79 98.92 ± 0.95 0.96 1.0 98.82 ± 1.12 1.13 98.76 ± 1.2 1.22 2.0 98.90 ± 1.15 1.17 99.15 ± 0.73 0.74 Each result is the average of three separate determinations. Pharmaceutical preparations The studied drugs were assayed in their ampoules and the results were satisfactory as in Table V. The results of their assay were in good agreement with those of the comparison method (4). Table V. Assay Results for the Determination of the Studied Drugs in their Different Dosage Forms by Proposed and Comparison Methods Proposed method Comparison method (4) Dosage form Amount taken Amount found % recovery Amount taken Amount found % recovery 1-CIS ampoules (CIS 25 mg /2.5 mL) 0.1 0.102 102.0 40.0 40.134 100.34 0.5 0.494 98.80 50.0 49.731 99.46 0.7 0.704 100.57 60.0 60.134 100.22 Mean 100.46 100.01 ±S.D 1.10 0.36 t 1.000 F 9.336 2-Propofol ampoules (PRP 10 mg/mL) 0.5 0.502 100.4 40.0 40.279 100.70 1.0 0.997 99.7 50.0 49.441 98.88 2.0 2.001 100.05 60.0 60.279 100.47 Mean 100.05 100.02 ±S.D 0.23 0.75 t 0.146 F 10.633 Proposed method Comparison method (4) Dosage form Amount taken Amount found % recovery Amount taken Amount found % recovery 1-CIS ampoules (CIS 25 mg /2.5 mL) 0.1 0.102 102.0 40.0 40.134 100.34 0.5 0.494 98.80 50.0 49.731 99.46 0.7 0.704 100.57 60.0 60.134 100.22 Mean 100.46 100.01 ±S.D 1.10 0.36 t 1.000 F 9.336 2-Propofol ampoules (PRP 10 mg/mL) 0.5 0.502 100.4 40.0 40.279 100.70 1.0 0.997 99.7 50.0 49.441 98.88 2.0 2.001 100.05 60.0 60.279 100.47 Mean 100.05 100.02 ±S.D 0.23 0.75 t 0.146 F 10.633 The value of tabulated t and F are 2.78 and 19.00, respectively at P = 0.05 (31). Each result is the average of three separate determinations. Table V. Assay Results for the Determination of the Studied Drugs in their Different Dosage Forms by Proposed and Comparison Methods Proposed method Comparison method (4) Dosage form Amount taken Amount found % recovery Amount taken Amount found % recovery 1-CIS ampoules (CIS 25 mg /2.5 mL) 0.1 0.102 102.0 40.0 40.134 100.34 0.5 0.494 98.80 50.0 49.731 99.46 0.7 0.704 100.57 60.0 60.134 100.22 Mean 100.46 100.01 ±S.D 1.10 0.36 t 1.000 F 9.336 2-Propofol ampoules (PRP 10 mg/mL) 0.5 0.502 100.4 40.0 40.279 100.70 1.0 0.997 99.7 50.0 49.441 98.88 2.0 2.001 100.05 60.0 60.279 100.47 Mean 100.05 100.02 ±S.D 0.23 0.75 t 0.146 F 10.633 Proposed method Comparison method (4) Dosage form Amount taken Amount found % recovery Amount taken Amount found % recovery 1-CIS ampoules (CIS 25 mg /2.5 mL) 0.1 0.102 102.0 40.0 40.134 100.34 0.5 0.494 98.80 50.0 49.731 99.46 0.7 0.704 100.57 60.0 60.134 100.22 Mean 100.46 100.01 ±S.D 1.10 0.36 t 1.000 F 9.336 2-Propofol ampoules (PRP 10 mg/mL) 0.5 0.502 100.4 40.0 40.279 100.70 1.0 0.997 99.7 50.0 49.441 98.88 2.0 2.001 100.05 60.0 60.279 100.47 Mean 100.05 100.02 ±S.D 0.23 0.75 t 0.146 F 10.633 The value of tabulated t and F are 2.78 and 19.00, respectively at P = 0.05 (31). Each result is the average of three separate determinations. Biological fluids Known amounts of the CIS and PRP were added to aliquots of human plasma samples to obtain various final concentrations ranging from 0.01 to 0.05 μg/mL and from 0.1 to 0.5 respectively. These values were previously reported for the two drugs in human plasma (33, 34). The sensitivity of the proposed method is high enough to determine CIS and PRP in spiked human plasma samples. Precipitation with methanol was adopted for the estimation of CIS and PRP in plasma samples. Under the previously described experimental conditions, a linear relationship was established by plotting the peak area against the drug concentration in μg/mL. The following equations represent the linear regression analysis: P=1316083.600+115944360.000C(r=0.9979)forCISP=−14380.200+974449.000C(r=0.9999)forPRP The high values of the correlation coefficients (r) point out to the good linearity of the plasma calibration graphs. The results for the assay in plasma are summarized in Table VI. The proposed method was applied for the determination of CIS and PRP in spiked human plasma over the concentration range of 0.01–0.05 and 0.10–0.50 μg/mL, respectively. Figure 4 shows representative chromatogram for spiked human plasma sample. Table VI. Results for the Determination of the CIS and PRP in Spiked Human Plasma Samples Using the Proposed Method Matrix Amount added (μg/mL) Amount found (μg/mL) % Recovery CIS PRP CIS PRP CIS PRP Spiked human plasma 0.01 0.10 0.0105 0.098 105.00 98.00 0.02 0.20 0.019 0.204 95.00 102.00 0.03 0.40 0.031 0.397 103.33 99.25 0.05 0.50 0.049 0.501 98.00 100.20 X¯ 100.33 99.86 ±SD ±4.64 ±1.69 % RSD 4.63 1.69 Matrix Amount added (μg/mL) Amount found (μg/mL) % Recovery CIS PRP CIS PRP CIS PRP Spiked human plasma 0.01 0.10 0.0105 0.098 105.00 98.00 0.02 0.20 0.019 0.204 95.00 102.00 0.03 0.40 0.031 0.397 103.33 99.25 0.05 0.50 0.049 0.501 98.00 100.20 X¯ 100.33 99.86 ±SD ±4.64 ±1.69 % RSD 4.63 1.69 Table VI. Results for the Determination of the CIS and PRP in Spiked Human Plasma Samples Using the Proposed Method Matrix Amount added (μg/mL) Amount found (μg/mL) % Recovery CIS PRP CIS PRP CIS PRP Spiked human plasma 0.01 0.10 0.0105 0.098 105.00 98.00 0.02 0.20 0.019 0.204 95.00 102.00 0.03 0.40 0.031 0.397 103.33 99.25 0.05 0.50 0.049 0.501 98.00 100.20 X¯ 100.33 99.86 ±SD ±4.64 ±1.69 % RSD 4.63 1.69 Matrix Amount added (μg/mL) Amount found (μg/mL) % Recovery CIS PRP CIS PRP CIS PRP Spiked human plasma 0.01 0.10 0.0105 0.098 105.00 98.00 0.02 0.20 0.019 0.204 95.00 102.00 0.03 0.40 0.031 0.397 103.33 99.25 0.05 0.50 0.049 0.501 98.00 100.20 X¯ 100.33 99.86 ±SD ±4.64 ±1.69 % RSD 4.63 1.69 Figure 4. View largeDownload slide Typical chromatogram of the studied drugs in spiked human plasma under the described chromatographic conditions: (A) blank plasma. (B) Plasma sample spiked with CIS (0.05 μg/mL) and PRP (0.5 μg/mL). Figure 4. View largeDownload slide Typical chromatogram of the studied drugs in spiked human plasma under the described chromatographic conditions: (A) blank plasma. (B) Plasma sample spiked with CIS (0.05 μg/mL) and PRP (0.5 μg/mL). Discussion There is evidence that using CIS and PRP during surgical operations is frequent due to their synergistic action. CIS is neuromuscular blocking agent. PRP is a short-acting anesthetic which give the best recovery for short time operations that are less than 30 min (35). The literature reported an HPLC method using UV detection for the separation of the two drugs (4). The proposed method has several advantages over the reported one: interestingly, using HPLC coupled with fluorescence detection resulted in increased sensitivity. In addition using monolithic column provided short time of analysis (<5 min). Moreover; the suggested procedure was used successfully to determine the CIS and PRP simultaneously in spiked human plasma (in vitro). Conclusion Referring to the importance of two drugs (CIS and PRP) which are co-administered during surgery, new HPLC methodology has been developed for their simultaneous determination in spiked human plasma. The assay procedure involved the use of HPLC coupled with fluorescence detection. The proposed method is highly sensitive, as down to 2.0 and 30 ng/mL of CIS and PRP could be detected respectively. The monolithic column permits the separation to be performed in <5 min. The method also utilized to the estimation of both compounds in their dosage forms. References 1 Sweetman, S. Martindale . ; (The complete drug reference). In Electronic version . The Pharmaceutical Press , London , ( 2009 ). 2 The United States Pharmacopoeia 34 . , The National Formulary 29; the US Pharmacopoeial Convention: Rockville, MD, ( 2011 ). 3 The British Pharmacopoeia . , Her Magesty’s Stationary Office: London, 2015 ; Vol. II. 4 Zhang , H. , Wang , P. , Bartlett , M.G. , Stewart , J.T. ; HPLC determination of cisatracurium besylate and propofol mixtures with LC-MS identification of degradation products ; Journal of Pharmaceutical and Biomedical Analysis , ( 1998 ); 16 ( 7 ): 1241 – 1249 . Google Scholar CrossRef Search ADS PubMed 5 Bryant , B.J. , James , C.D. , Ryan Cook , D. , Croft Harrelson , J.;. ; High performance liquid chromatographic assay for cisatracurium and its metabolites in human urine ; Journal of Liquid Chromatography & Related Technologies , ( 1997 ); 20 ( 13 ): 2041 – 2051 . Google Scholar CrossRef Search ADS 6 Gao , J. , Yang , T. , Ye , M. , Zhang , X. , Tian , G. , Zhen , Q. , et al. . ; High-performance liquid chromatography assay with programmed flow elution for cisatracurium in human plasma: Application to pharmacokinetics in infants and children ; Journal of Chromatography, B: Biomedical Sciences and Applications , ( 2014 ); 955-956 : 58 – 63 . 7 Li , J. , Chen , B. , Yang , W , Zhang , Y. ; Determination of cisatracurium in human plasma by iron-pair HPLC with fluorescence detection ; Chinese Journal of Pharmaceutical Analysis , ( 2011 ); 31 : 713 – 716 . 8 Błazewicz , A. , Fijałek , Z. , Warowna-Grześkiewicz , M. , Jadach , M. ; Determination of atracurium, cisatracurium and mivacurium with their impurities in pharmaceutical preparations by liquid chromatography with charged aerosol detection ; Journal of Chromatography A , ( 2010 ); 1217 ( 8 ): 1266 – 1272 . Google Scholar CrossRef Search ADS PubMed 9 Rut , F. , Miguel , A.B. , Manuel , C.N. , Juan , C.J. , Nez , A.G. ; Spectrofluorimetric determination of cisatracurium and mivacurium in spiked human serum and pharmaceuticals ; Talanta , ( 1999 ); 49 : 881 – 887 . Google Scholar CrossRef Search ADS PubMed 10 Fernandez , T. , Callejon , M. , Jimenez , S. , Bello Lopez , M.A. , Perez , A.G. ; Electrochemical oxidation of cisatracurium on carbon paste electrode and its analytical applications ; Talanta , ( 2001 ); 53 : 1179 – 1185 . Google Scholar CrossRef Search ADS PubMed 11 Ming , Z. , Jieying , G. , Xiaoqing , Z. , Yue , C. , Zimian , F. , Min , D. ; Capillary electrophoresis with electrochemiluminescence detection for the simultaneous determination of cisatracurium besylate and its degradation products in pharmaceutical preprations: electrodriven separations ; Journal of Separation Science , ( 2015 ); 38 ( 13 ): 2332 – 2339 . Google Scholar CrossRef Search ADS PubMed 12 Pissinis , D. , Sereno , L.E. , Marioli , J.M. ; Multi-wavelength spectrophotometric determination of propofol acidity constant in different acetonitrile-water mixture ; Journal of Brazilian Chemical Society , ( 2005 ); 16 ( 5 ): 1054 – 1060 . Google Scholar CrossRef Search ADS 13 VanaŠ , R. , Célia , G. , Amorim , H.S. , Maria , C.B.M. , Burkhard , H. , Alberto , N.A. , et al. . ; Fully automated analytical procedure for propofol determination by sequential injection technique with spectrophotometric and fluorimetric detections ; Talanta , ( 2014 ); 118 : 104 – 110 . Google Scholar CrossRef Search ADS PubMed 14 Dawidowicz , A.L. , Fijalkowska , A. ; Determination of propofol in blood by HPLC. Comparison of the extraction and precipitation methods ; Journal of Chromatographic Science , ( 1995 ); 33 ( 7 ): 372 – 382 . Google Scholar CrossRef Search ADS 15 King , D.T. , Stewart , J.T. , Venkateshwaran , T.G. ; HPLC determination of propofol-thiopental sodium and propofol-ondansetron mixtures ; Journal of Liquid Chromatography and Related Technology , ( 1996 ); 19 ( 14 ): 2285 – 2294 . Google Scholar CrossRef Search ADS 16 Vishwanathan , K. , Stewart , J.T. ; HPLC determination of a propofol and remifentanil mixture ; Journal of Liquid Chromatography and Related Technology , ( 1999 ); 22 ( 6 ): 923 – 931 . Google Scholar CrossRef Search ADS 17 Yeganeh , M.H. , Ramzan , I. ; Determination of propofol in rat whole blood and plasma by high- performance liquid chromatography ; Journal of Chromatography B , ( 1997 ); 691 ( 2 ): 478 – 482 . Google Scholar CrossRef Search ADS 18 Kwak , J.H. , Hye , K.K. , Sanggli , C. , Sangwhau , L. , Jae , S.P. ; Determination of propofol glucouronide from hair sample by using mixed mode anion exchange cartridge and liquid chromatography mass spectrometry ; Journal of Chromatography B , ( 2016 ); 1015-1016 : 209 – 213 . Google Scholar CrossRef Search ADS 19 Fabio , V. , Giovanni , S. , Mathia , F. , Alessia , F. , Francesco , M. , Elisabetla , B. ; LC-MS/MS and GC-MS methods in propofol detection. Evaluation of two analytical procedures ; Forensic Science International , ( 2015 ); 266 : 1 – 6 . 20 Lambert , K.S. , Jorgen , B.H. ; Simultaneous determination of propfol and its glucouronide in whole blood by liquid chromatography electrospray tandem mass spectrometry and the influence of sample storage conditions on the reliability of the test results ; Journal of Pharmaceutical and Biomedical Analysis , ( 2015 ); 109 : 158 – 163 . Google Scholar CrossRef Search ADS PubMed 21 Kim , H.S. , Cheong , J.C. , Lee , J.I. , In , M.K. ; Rapid and sensitive determination of propofol glucouronide in hair by liquid chromatography and tandem mass spectrometry ; Journal of Pharmaceutical and Biomedical Analysis , ( 2013 ); 85 : 33 – 39 . Google Scholar CrossRef Search ADS PubMed 22 Mariska , Y.M.P. , Hiltjo , K. , Ben , G. , Joukje , V.D.N. , Catherijne , A.J.K. , Donald , R.A.U. ; Gas chromatography mass spectrometric assay for propofol in cerebrospinal fluid of traumatic brain patients ; Journal of Chromatography B , ( 2007 ); 852 : 635 – 639 . Google Scholar CrossRef Search ADS 23 Francis , B. , Sarah , A.G. , Andrew , W. , Jean , F.M. , Pascal , V. ; Development of a rapid and sensitive LC-ESI/MS/MS assay for the quantification of propofol using a simple offline dansyl chloride derivatization reaction to enhance signal intensity ; Journal of Pharmaceutical and Biomedical Analysis , ( 2005 ); 39 : 411 – 417 . Google Scholar CrossRef Search ADS PubMed 24 Knibbe , C.A.J. , Koster , V.S. , Deneer , V.H.M. , Stuurman , R.M. , Kuks , P.F.M. , Lange , R. ; Determination of propofol in low-volume samples by high-performance liquid chromatography with fluorescence detection ; Journal of Chromatography B , ( 1998 ); 706 : 305 – 310 . Google Scholar CrossRef Search ADS 25 Tadashi , N. , Rie , S. , Yuko , T. , Hideko , K. , Teruo , O. , Takako , M.N. ; Aqueous chromatographic system for the quantification of propofol in biological fluids using a temperature responsive polymer modified stationary phase ; Journal of Chromatography A , ( 2009 ); 1216 : 7427 – 7432 . Google Scholar CrossRef Search ADS PubMed 26 Sun , Y.L. , Na-Hyun , P. , Eun-Kyung , J. , Jae-Woo , W. , Chang-Ju , K. , MoonKuo , I. , et al. . ; Comparison of GC/MS and LC/MS methods for the analysis of propfol and its metabolites in urine ; Journal of Chromatography B , ( 2012 ); 900 : 1 – 10 . Google Scholar CrossRef Search ADS 27 Si-Cheng , L. , Guang-Bo , G. , Hui-Xin , L. , Hai-Tao , S. , Hong , W. , Zhong-Ze , F. , et al. . ; Determination of propofol UDP-glucuronosyltransferase (UGT) activities in hepatic microsomes from different species by UFLC-ESI-MS ; Journal of Pharmaceutical and Biomedical Analysis , ( 2011 ); 54 : 236 – 241 . Google Scholar CrossRef Search ADS PubMed 28 Cohen , S. , Lhuillier , F. , Mouloua , Y. , Vignal , B. , Favetta , P. , Guitton , J. ; Quantitative measurement of propofol and in main glucuroconjugate metabolites in human plasma using solid phase extraction-liquid chromatography-tandem mass spectrometry ; Journal of Chromatography B , ( 2007 ); 854 : 165 – 172 . Google Scholar CrossRef Search ADS 29 Jan , L. , Fernando , G. , Fracine , K. , Edward , C. , Erno , L. ; Electrochemical quantification of 2,6-diisopropylphenol(propofol) ; Analytica Chimica Acta , ( 2011 ); 704 : 63 – 67 . Google Scholar CrossRef Search ADS PubMed 30 Nathan , N. , Bonada , G. , Feiss , P. ; Potentiation of atracurium by pancuronium during propofol-fentanyl-N2O anesthesia ; Acta Anaesthesiol Belgica , ( 1996 ); 47 ( 4 ): 187 – 193 . 31 Miller , J.N. , Miller , J.C. ; Statistics and Chemometrics for Analytical Chemistry, Harlow , 5th ed. . Pearson Education Limited , Edinburgh Gate, Harlow , ( 2005 ); pp. 39 – 73 . 107-149, 256. 32 ICH Harmonized Tripartite Guidelines . , Validation of Analytical Procedures: Text and Methodology, Q2(R1), Current Step 4 Version, Parent Guidelines on Methodology Dated November 6; 1996, Incorporated in November ( 2005 ). 33 Moffat , A.C. , Osselton , M.D. , Widdop , B. , Galichet , L.Y. ; Clark’s Analysis of Drugs and Poisons in Pharmaceuticals, Body Fluids and Postmortem Material , Vol II , 4th ed. . The Pharmaceutical Press , London , ( 2011 ); pp. 1591 – 1698 . 34 Zhang , T. , Cui , Y. ; Determination of propofol in human plasma by microemulsion liquid chromatography; Chinese ; Journal of Chromatography , ( 2011 ); 29 ( 8 ): 768 – 772 . 35 Aronson , J.K. ; Meyler’s Side Effects of Drugs Used in Anesthesia , 198 . Elsevier Science & Technology , Oxford , ( 2009 ); pp. 63 – 78 . © 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)

Journal

Journal of Chromatographic ScienceOxford University Press

Published: Apr 9, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off