Submission number: 235 Proceedings of RK University’s first international Conference on Research & Entrepreneurship (Jan. 5 th & Jan. 6th, 2016) ISBN: 978-93-5254-061-7(Proceedings available for download at rku.ac.in/icre)
RK University’s First International Conference on Research & Entrepreneurship (ICRE 2016)
Preformulation Studies & Preparation Of Cyclophosphamide Loaded Solid Lipid Nano Particles Sankha Bhattacharya1,*, Bhupendra Prajapati2, and Maulik Mehta3 1
PhD scholar, School of Pharmacy, RK University, Rajkot-Bhavnagar Highway, Rajkot-360020, Gujarat, India & Assistant Professor, B. Pharmacy college-Rampura, Godhra, Gujarat-389001, India. 2
Associate Professor, Dept. of Pharmaceutical Technology, Ganpat University, Kherva, Mehsana, Gujarat384012, India. 3
Principal at B. Pharmacy college-Rampura, Godhra, Gujarat -389001.
*
Corresponding author: Mr. Sankha Bhattacharya (
[email protected])
--------------------------------------------------------------------------------------------------------------ABSTRACT: Cyclophosphamide is a chemotherapeutic drug belongs to nitrogen mustard alkylating agents. It is derived from oxazophorines group. It has good anti-cancer activity. Many injectable forms of cyclophosphamide are available, but all has poor bioavailability and nonspecific targeting problems. It was attempted to formulate cyclophosphamide in the form of cationic solid lipid nanoparticles. Solid lipid nanoparticles were obtained by adsorption of lipid dispersion in the organic phase and diffusion in the aqueous phase of the high-speed Homogenizer technique at 30000 RPM. Eventually, pre-formulation tests are carried out to escalate compatibility of drug and excipient. We performed FTIR, DSC, XRD studies. No significant changes observed in drug and solid lipid compatibility. Melting point, partition coefficient, UV, HPLC studies shows satisfactory results. After preformulation study cyclophosphamide loaded solid lipid nanoparticles was prepared. It is concluded that solid lipid nanoparticles of cyclophosphamide could be formulated with the selected lipids. SUMMARY: Preformulation studies and preparation of cyclophosphamide loaded solid lipid nanoparticles. Keywords: Cyclophosphamide, Solid lipid nanoparticles, Pre-formulation studies, DSC, FTIR, XRD. ----------------------------------------------------------------------------------------------------------------------------- --------1. INTRODUCTION: Glioblastoma multiforme (GBM) one of the most devastating brain carcinoma, it has a habitual recurrence with narrow prognosis (1). This physiological condition got recently much more medical attention, as the number of deaths increasing rapidly by brain carcinoma. For a considerable period of time chemotherapy is the last choice in GBM, but the systematic administration of chemotherapeutic drugs causes savior non-selective cytotoxicity on normal cells, secondary alopecia is also observed on the diseased. So specific
targeting to the cancerous cell become a paramount importance in cancer treatment (2). Nanoparticular drug delivery systems emerging as an excellent approach to circumvent all those associated problems with chemotherapy, as nanomedicines are below 1µm and because of this property, easy endocytosis
through the
Blood Brain Barrier (BBB), enhance cell internalization and delivery of drugs to the intracellular matrix are possible (3). Apart from it, conjugation with biodegradable polymers, anti -epidermal growth factor, folic acids, antibody along with core drugs, enhance cellular uptake and selectivity. With up gradation in nano delivery systems, Solid Lipid Nano Particles (SLNs) becoming an emerging nanocarrier for chemotherapeutic drugs because of its high cellular uptake and rapid clearance. SLNs prepared from lipids such as Triglycerides (Trilaurin™, Tristearin™, Dynasan122™), Acylglycerides (Glyceryl monostearate, Glyceryl Palmitostearate) Fatty acids (Stearic acid, Palmitic acid, Behenic acid, Waxes (Cetyl alcohol, Beeswax, cholesterol, Emulsifying wax NF), Hydrogenated soybean oil (Lipo ™ or Sterotex™) (4). Recent studies confirmed (Giulia Fulci et al) Cyclophosphamide a synthetically modified antineoplastic drug obtained from nitrogen muster, enhanced glioma virotherapy by inhibiting the inner immune response. In rat glioma model (5) cyclophosphamide enhance the natural killer cells, microglia associated with macrophages of CD68+ & CD163+, INF-ϒ. This research accelerates the need for extensive research on cyclophosphamide as a nanocarrier drug. In this research, we carried out our pre-formulation studies on the model drug as cyclophosphamide. We attempted to formulate Solid lipid nanoparticles of cyclophosphamide using a different ratio of Glyceryl monostearate and citric acid as a lipid, Soya lecithin as co-surfactant, Brij 78, Poloxamer 188, Tween 80 as a surfactant, HPLC water as an aqueous solvent, Dichloromethane, methanol (acetone free) as an organic solvent. The formulation was prepared by solvent diffusion and dispersion technique (6). 2. MATERIALS: Cyclophosphamide model drug was obtained as a gift sample from Emcure Pharmaceuticals LTD. Ahmadabad, India. Glycerol monostearate (product No: 17145) purchased from Astron chemicals, Ahmadabad, India. Soya lecithin (Product no: 88993) purchased from Astron chemical, Ahmadabad, India. Poloxamer 188 purchased from Balaji drugs Surat, India. Dichloromethane from Chem dyes corporation–Rajkot. Brij 78 obtained from Chem dyes corporation–Rajkot. Tween-80 procured from fine star industry. Carbon tetrachloride procured from Chem dyes corporation–Rajkot. Methanol (acetone free) was purchased from Chem dyes corporation–Rajkot. Polyvinyl alcohol and lactose monohydrate from Astron chemicals Ahmadabad, India. Ultipor N56 Nylon 6, 6 membrane filter-0.45mm, procured from Pall life science. Phosphate buffer from J&K Scientific LTD, 1L HPLC grade water from Fisher scientific Ltd.100ml Acetonitrile was purchased from Imperial Chemical. Ammonium molybdate, Ferric chloride solution, Stannous chloride are gift sample from RK University. 3. METHODS: 3.1. UV analysis: 3.1.1. Estimation of cyclophosphamide by UV spectroscopy (7): 3.1.2. Apparatus: Measurement was carried out by using SHIMADZU-1880UV-VIS Spectrophotometer at 722nm. 3.1.3. Sample: Cyclophosphamide model drug was obtained as a gift sample from Emcure Pharmaceuticals, Ahmadabad, India.
3.1.4. Materials and reagents for standard absorbance and linearity: All the chemicals used were of analytical grade. All the solutions were prepared freshly and the deionized water is used throughout the experiment. 3.1.4.1. Ammonium molybdate (0.005M): Weighed exactly 0.61795g (± 0.0001g) of ammonium molybdate in 100 ml volumetric flask it was dissolved and diluted to the mark using deionised water. 3.1.4.2. Ferric chloride solution (0.01M): Weighed exactly 0.22560g (±0.0001g) of ferric chloride in 100 ml volumetric flask it was dissolved and then diluted to the mark using double distilled alcohol. 3.1.4.3. Stannous chloride (0.01M): Weighed exactly 0.16221g (±0.0001g) of stannous chloride in 100 ml volumetric flask it was dissolved and diluted to the mark with deionized water. Pharmaceutical grade Cyclophosphamide (CP) claimed to be 99.7 % pure was received from the Emcure Pharmaceuticals, Ahmadabad, India. Which is used as a reference standard for the analysis without any further purification? 3.1.5. Standard drug solution: A stock solution of CP (1 mg/ml) was prepared by using 10 mg of the reference standard drug in a 10 ml volumetric flask, added 5 ml methanol to dissolve the content and brought up to the mark with methanol, mixed thoroughly for homogeneity. Working solution of lower concentration (100μg/ml) was prepared by further dilution of the above standard stock solution with methanol. 3.1.6. Linearity studies: For linearity studies we selected a different set of concentration, but the best curve fits between 20- 45 µg/ml of standard cyclophosphamide.At first six 10ml volumetric flask were taken & prepared 100µg/ml working standard first, from that 2ml,2.5ml,3ml,3.5ml,4ml,4.5ml were withdrawn and transferred into six 10 ml volumetric flask, then 1ml ferric chloride were added and all the content were kept in warm condition for 5 minutes, then contents were filtered and 2ml ammonium molybdate followed by 2ml of hot stannous chloride were added, slowly formation of blue colour phosphomolybdate complex were seen, then all the volumetric flasks were filled up to 10ml with double distilled water and kept at room temperature for cooling. The experiments were repeated for three times, and all eighteen volumetric flask content were measured at 722nm using reagent as a blank The average absorbance’s were selected for standard curve(figure1,table1).The calibration curve was plotted against absorbance(nm) versus concentration(µg/ml). 3.1.7. Determination of the absorption maxima (λmax): To determine the λmax of the colored species, 1 ml of 100 μg/ml of the CP was added to a test tube and 1 ml of ferric chloride solution, heated the contents for 5 min. Filtered the contents to 10 ml standard volumetric flask, add 2 ml of ammonium molybdate solution followed by adding 2 ml of hot stones chloride solution resulting in the formation of a deep blue colored phosphomolybdate complex then the flasks are allowed to cool to room temperature and the solution made up to the mark with water. The colored species were measured against reagent blank in the range of 400 NM at 800 NM. The λmax of the complex was found to be 722 mm. The absorption spectrum of the proposed method was shown in figure2. Under the experimental conditions, each reagent blank showed a negligible absorbance at the corresponding λmax. 3.2. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY STUDIES ON CYCLOPHOSPHAMIDE: 3.2.1 Chromatography condition (8): A chromatographic system Dionex ultimate 300, consisting of a quaternary solvent delivery pump, and autoinjector, column oven and UV detector, A thermal scientific column ODS, a stationary phase with particle size 5µ, and pore size 100A was used. The column is end capped and carbon
content of 11%, the flow rate was maintained 1.5ml/min (Table2).Blank and standard drug estimated by HPLC (figure 3&4, table 2, 3, 4, 5, 6) 3.2.2. Preparation of stock solution: 50mg standard cyclophosphamide API was accurately weighed and transferred into the 50ml volumetric flask, later dissolved with diluent (HPLC water50ml: acetonitrile 50ml %v/v) to produce 1000µg/ml. Further, withdraw 1ml of above solution and transfer in the 100ml volumetric flask.Add above-mentioned diluent to produce 100ml.This will give 10µg/ml solution. 3.3. FOURIER TRANSFORM INFRARED SPECTROSCOPY: The pure and dryad cyclophosphamide were triturated with powdered potassium bromide in a proportion of 1:200.The triturated amount should be taken such a way that, it would spread the area of the disk 5-15 g per 2 mm. By using high pressure or vacuum pressure a portion of the mixture was inserted. In suitable holder mount the resultant disk. Sometimes impurities in holder disk, inadequate or excessive grinding, and moisture in triturate causes unpleasable disks.After visual all inspection the disk to be placed. IR spectra run within 4000-400cm-1 range.Same way IR spectra of Drug with a physical mixture of Solid lipids; GSM, Stearic acids were performed.The experiments were carried out in SHIMADZU–FTIR-8400s model (figure 5, 6, 7 and table 7, 8). 3.4 DIFFERENTIAL SCANNING CALORIMETRY (DSC): The interaction between cyclophosphamide, GMS, stearic acid, soya lecithin has been checked in the solid state by SHIMADZU–DSC-60 Plus instrument. The chance of complex formation within the substances was marginal. By measuring the thermal behavior of all substances with the drug showed in the phase diagram, indicates there was no interaction between the drug and excipients.The DSC curve of cyclophosphamide showed a melting endodermal at 51.20OC.The normal melting point of cyclophosphamide as per monograph is in between 45-53 O C which is an incompatible range. The drug and physical mixture were prepared from GMS and Stearic acid, both the compound has a melting point between 50-61OC. The drug and physical mixture endotherm show pick at 52.88 OC, and in 123.23OC, 161.25OC respectively, indicated no significant difference in melting endotherm with respect to melting point of an individual ingredient(figure 8,9).This proved the computability of the drug with other two ingredients(9). 3.5. MELTING POINT DETERMINATION: The fully dryad, free flowing powder of cyclophosphamide was loaded into a capillary tube, the capillary tube must be sealed in at one end before use. The powder must get settled in the bottom of the capillary tube against a hard surface. It was cross checked that the optimum 3-4mm of powder height in capillary could provide reproducible and accurate results. Finally, in Buchi ® B-540 melting point apparatus slot the sample capillary was inserted.The temperature at which cyclophosphamide state’s meeting was recorded. It was found to be 530C. 3.6. CYCLOPHOSPHAMIDE POWDER X-RAY DIFFRACTION (PXRD): Dataset Name:
STD drug-cyp
File name:
D:\XRD Data\Demo\Sankha 7 12 15\Std drug.xrdml
Measurement Date / Time:
21-08-2012 06:34:35
Operator:
XPERT
Raw Data Origin:
XRD measurement (*.XRDML)
Scan Axis:
Gonio
Start Position [°2Th.]:
5.0042
End Position [°2Th.]:
79.9882
Step Size [°2Th.]:
0.0080
Scan Step Time [s]:
5.7150
Scan Type:
Continuous
PSD Mode:
Scanning
PSD Length [°2Th.]:
2.12
Divergence Slit Type:
Fixed
Divergence Slit Size [°]:
0.8709
Specimen Length [mm]:
10.00
Measurement Temperature [°C]:
25.00
Anode Material:
Cu
K-Alpha1 [Å]:
1.54060
Generator Settings:
40 mA, 45 kV
Goniometer Radius [mm]: 240.00 Incident Beam Monochromator: No The diffraction pattern of the Plain drug showed characteristic high-intensity diffraction peaks at 6.66, 14.67, 17.47, 25.36, 28.88, 29.75, 33.60, and 35.31 of 2theta.This indicates that the pure drug of cyclophosphamide is in crystalline form (figure 10, table 9). 3.7. DETERMINATION OF PARTITION COEFFICIENT OF CYCLOPHOSPHAMIDE DRUG AND WITH GLYCERYL MONOSTEARATE (LIPID): By taking known amount of drug in separating funnel, add an equal amount of aqueous phase (double distilled water) and organic phase as chloroform. Shake vigorously and releases pressure in the frequent interval, shake for 10 minutes and allow the layer to separate.Calculate the amount of drug in both layers using UV analysis. 3.7.1. Observation: Pka = Drug concentration of organic layer / Drug concentration in aqueous = 0.63 3.8. Partition coefficient of drug in Glyceryl monostearate (Lipid) and Phosphate buffer p H 7.4: 10 mg of Cyclophosphamide was added in a mixture of melted lipid, Glyceryl monostearate (Melting point >55ºC1g) and 10ml of Phosphate buffer of 37ºC (Fisher Scientific) and shaken for 30 minutes in a mechanical shaker (Remi, Mumbai), using a moderate hot water bath maintained 10ºC above the melting point of the lipid. The aqueous phase of the above mixture was separated from the lipid by centrifugation at a speed of 6600 RPM for 20 minutes at -57ºC temperature using Refrigerator centrifuge (EMTEK Instrument India).The clear suspension was suitably diluted with p H 7.4 phosphate buffer and the cyclophosphamide content was quantified using UVvisible
spectrophotometer (Elite) at 722 nm against a solvent blank.The partition coefficient of
Cyclophosphamide in lipid/ p H 7.4 PB was calculated using equation.Partition coefficient = CL/CA . CL is the amount of cyclophosphamide in lipid and CA is the amount of cyclophosphamide in pH7.4 PB.
3.8.1. Observation: The partition coefficient in GSM was found to be 1.45 3.9. PERMEABILITY COEFFICIENT: The permeability coefficient of drug was calculated by using Potts and Guy equation as mentioned bellow: LogKp = -6.3 + 0.71X log Ko/w -0.0061 X Molecular weight Where, Log Kp =Permeability coefficient Ko/w = Partition coefficient. Log Kp value of cyclophosphamide was found to be -8.144. Were Molecular weight of cyclophosphamide is 279.10 & partition coefficient is 0.63. LogKp value of cyclophosphamide with GMS was found to be -10.086, where the molecular weight of cyclophosphamide and GMS combined; that is 637.7. Where Ko/w was 1.45. LogP = In (Ko/w) =-0.46(Cyclophosphamide) Log P = In (Ko/w) = 0.371 (Cyclophosphamide +GMS) 3.10. SOLUBILITY ANALYSIS: 10 mg of cyclophosphamide was dissolved in 10 ml of different solvent i.e., water, carbon tetra chloride, methanol (acetone free), dichloride methane, chloroform, glacial acetic acid, and ethanol. Solubility were tested as per physical appearance.
4. SCHEMATIC REPRESENTATIONS ON PREPARATIONS OF CATIONIC SOLID LIPID NANOPARTICLES OF CYCLOPHOSPHAMIDE SLNs prepared by melt dispersion technique and ultrasonication method. : Formulation of Cationic solid lipid Nanoparticles:
Soya lecithin (0.75%w/v) +GMS: SA-30mg 15ml HPLC water +Cyclophosphamide
+PVA (0.02%) dissolved in
(15mg) + Ploxamer 188(0.35%w/v)
15ml Methanol (acetone free)
+Lactose monohydrate (0.08 %W/V)
In hot plate with Magnetic stirrer maintaining 60ºC Temperature.
20 minutes ultra-Sonication Dispersion with
Brij 78 +Tween 80 (0.35: 0.15 %V/V)
constant stirring Cetrimid (0.15% W/V) [Formation of lipid phase]
[Formation of aqueous phase]
Maintain temperature at 0-5ºC
[Aqueous phase is dispersed in lipid phase with constant stirring using High-pressure homogenizer at 30,000rpm speed for 25minutes]
[Ultra-sonicate for 10 minutes]
[Colloidal SLNs light bluish dispersion formed]
[Filter in 0.22-micrometer membrane filter and lyophilize]
[Cationic–SLNs of cyclophosphamide]
5.RESULT AND DISCUSSION: 5.1. UV analysis of cyclophosphamide: Preformulation usually performed to select a perfect drug and ingredients for the formulation.Cyclophosphamide produces a maximum absorbance at 722nm.
5.2. A standard curve of cyclophosphamide: Cyclophosphamide does not possess linearity bellow 20µg/ml concentration.So concentration was selected between 20-45 µg/ml.For linearity, we prepared 3 different sets of samples ranging from 20-45µg/ml.Absorbance was taken at 722nm after complete blue color phosphomolybdate complex formation in 10ml volumetric flux. Mean absorbance was selected for linearity.The slope was found to be 0.0344 and regression coefficient was found to be 0.9991. 5.3. High-Performance liquid chromatography: Reverse phase HPLC of cyclophosphamide was performed using solvent as HPLC Water: Acetonitrile (50:50%v/v) and 0.05M Potassium dihydrogen orthophosphate (60): Acetonitrile (40) mixture as mobile phase .The flow rate was maintained inclined at 15ml/min. The chromatogram were obtained for blank at 1.23 & for standard 2.12. 5.3.1. An important observation during operation: Occasionally sample runs for 30 minutes, but during this period of this experiment no characteristic picks were obtained. The sample run time was extended up to 50 minutes. The blank is showing some extra pick because the observational range is the only 200nm, due to narrow rage, the possibility of showing other picks maximized apart from used solvent picks. 5.4. Fourier transform infrared spectroscopy: FTIR studies identified the drug, and also concluded that the drug has no interaction with a physical mixture including GMS, and Stearic acid. The principle picks were at 1100 cm-1, 2370cm-1, 1300 cm-1, 3100 cm-1, 1790 cm-1, 2750 cm-1, 3690cm-1, 1780 cm-1, 1250 cm-1.Each pick represents the functional group. It was concluded that there is no drug ingredient interaction and the drug is compatible with solid lipids. 5.5. Differential scanning calorimetry: The interaction between cyclophosphamide, GMS, Stearic acid, Soya lecithin has been checked in the solid state by SHIMADZU-DSC-60 Plus instrument. Thermographs showing that there was no interaction between drug and excipients. 5.6. Melting point: Melting point of cyclophosphamide by capillary method was found to be 53 oC. 5.7. XRD study on cyclophosphamide API: The diffraction pattern of the Plain drug showed characteristic highintensity diffraction peaks at 6.66, 14.67, 17.47, 25.36, 28.88, 29.75, 33.60, and 35.31 of 2theta.This indicates that the pure drug of cyclophosphamide is in crystalline form. 5.8. Determination of partition coefficient, permeability coefficient, and LogP value: Ko/w
LogKp
LogP
Drug
0.63
-8.144
-0.46
Drug+GMS
1.45
-10.086
0.371
As per pharmacopeias standard if Log P value is below zero, then the substance has injectable property, and if 03 then substance can consider for oral route administration.The drug-lipid partition coefficient is within the standard limits.
5.9. Solubility analysis: The drug is highly soluble in methanol (acetone free), fairly soluble in water, rapidly soluble in acetic acid, and slightly insoluble in dichloromethane. 6.CONCLUSION:Cyclophosphamide[N,N-bi's(2-chroethyl)-1,3,2-oxazaphosphinan-2-amine]maximum absorbance determined at 722nm using UV-Visible spectrophotometry (figure 1, 2). In linearity curve, the regression coefficient was found to be R2=0. 991 (figure 1, table 1). In Reverse phase, HPLC method retention time of the drug was tested, it was found to be 2.12 (Figure 3&4, Table 2,3,4,5,6). In FTIR studies, characteristic picks were observed at web number 1300,1985, 2370 cm-1(figure 5,6,7 and table 7,8 ).The drug was found to be compatible with solid lipids (GMS & Stearic acid). In DSC study drug shows picks at 52.20,180.84 OC and drug and physical mixture shows picks at 52.88,122.23
O
C, It was concluded that drug and physical mixture is
compatible with each other (figure 8,9).In XRD studies characteristic high-intensity diffraction peaks at 6.66, 14.67, 17.47, 25.36, 28.88, 29.75, 33.60, and 35.31 of 2theta (figure 10).This indicates that pure drug of cyclophosphamide is in crystalline form. The partition coefficient of drug and drug with GMS was found to be 0.63, 1.45 respectively.The melting point was found to be 53 O C. The solubility study indicate that drug is mostly soluble in methanol(acetone free) and in acetic acid (table 10). The SEM analysis of prepared solid lipid nanoparticle showing below 1µm ranged particles (figure11,12). Pre-formulation studies signify that cyclophosphamide is compatible with solid lipids, it is showing good retention of 2.12 in HPLC method, XRD, DSC, FTIR showed satisfactory results, the melting point is also within the limit, the drug is showing good partitioning with principle lipid of GMS. After solubility study, it was concluded that methanol (acetone free) could be the best alternative for preparing solid lipid nanoparticular pre-emulsion. Based on the preliminary results an attempt was made to prepare cationic solid lipid nanoparticles, initials results showing good stability and nano ranged particle formation. 7. FIGURES:
Cyclophosphamide linearity ABSORBANCE (NM)
2 y = 0.0344x - 0.0082 R² = 0.9991
1.5 1 0.5 0 0 -0.5
10
20
30
40
CONCENTRATION (ΜG/ML) Absorbance(nm)
Linear (Absorbance(nm))
Figure 1: Cyclophosphamide linearity curve
50
Figure 2: λmax of Cyclophosphamide observed at 722nm
Figure 3: HPLC chromatograph of blank
Figure 4: HPLC chromatograph of cyclophosphamide
Figure 5: IR spectra of standard cyclophosphamide drug
Figure 6: IR spectra of drug and physical mixture
Figure 7: Compatibility of Cyclophosphamide and physical mixture
DSC mW 1 8 0 .8 40 x1 0 C
10.00
0.00
-10.00
5 1 .2 00 x1 0 C
50.00
100.00
150.00 200.00 Temp [C]
250.00
300.00
Figure 8: DSC of cyclophosphamide pure drug
Thermal Analysis Result
DSC mW
10.00 0 161 .25x10 C
0.00 122 .230 x10 C
-10.00
-20.00
0 52.88x10 C
50.00
100.00
150.00 200.00 Temp [C]
250.00
300.00
Figure 9: Cyclophosphamide +Physical mixture (GMS+stearic acid)
Figure 10: XRD graph of Cyclophosphamide
Figure 11: Scanning electron microscopy photographs for cyclophosphamide solid lipid nano particles .a field containing different sized particles in a scale of 10µm using 100× magnification power
Figure 12: Scanning electron microscopy photographs for the cyclophosphamide solid lipid nanoparticles field containing different sized particles on a scale of 1µm using 1000× magnification power.
8. TABLES: Table1: Values of absorbance’s observed at 722nm Concentration (µg/ml) 0 20 25 30 35 40 45
Absorbance(nm) I
II
III
0 0.701 0.837 0.998 1.27 1.38 1.56
0 0.690 0.835 1.02 1.21 1.38 1.54
0 0.680 0.838 1.04 1.16 1.41 1.57
Mean absorbance (n=3)
0 0.690±0.003 0.836±0.001 1.01±0.002 1.18±0.005 1.39±0.002 1.55±0.003
Table 2: Method of analysis in HPLC-Reverse phase Parameters Instrument:
Description Thermo Fischer Scientific; RP-UHPLC
Instrument Model Detector:
Dionex Ultimate 3000 UV Detector
Column Flow rate Injection volume Column temperature
Thermo Scientific, ODS (250 mm x4.6 mm); Particle size 5µ 1.5 mL/min 10µL 30 ºC
Wavelength:
200 nm
Mobile phase
0.05 M Potassium dihydrogen orthophosphate (60): Acetonitrile (40)
Solvent mixture
Acetonitrile (50): Water (50)(%v/v)
Ua x Sd x p
x 100 Sa x Ud x 100
Where, Sd = Standard dilution Sa = Standard area Ud =Sample dilution Ua = Sample area P = Potency of Standard Drug
Table 3: HPLC –RP analysis of blank 01
BLANK
Sample Name: BLANK
Injection Volume (µL): 10.0
Vial Number: RE2
Channel: UV_VIS_1
The sample for : Mr. Sankha Bhattacharya; RK university Control Program: CYCLOPHOSPHAMIDE
Performed in: Supra drug and food testing research lab Operator: Juhi Bhatt
Quantif. Method: CYCLOPHOSPHAMIDE
Instrument ID: U3000
Recording Time: 04/12/15 11:21:50 Sequence: CYCLOPHOSPHAMIDE
Table 4: Peak result for blank in HPLC No.
Ret.Time
1 2
(min)
Peak Name
Height
0.97
n.a.
1.23
n.a.
(mAu)
Area (mAU*min)
Rel.Area (%)
101.053
326.507
35.23
33.291
600.357
64.77
926.864
100.00
Total:
Table 5: HPLC-RP analysis of standard drug of cyclophosphamide 02
STANDARD
Sample Name: STANDARD
Injection Volume (µL): 10.0
Vial Number: RE3
Channel: UV_VIS_1
The sample for: Mr. Sankha Bhattacharya; RK university
Performed in: Supra drug and food testing research lab
Control Program: CYCLOPHOSPHAMIDE
Operator: Juhi Bhatt
Quantif. Method: CYCLOPHOSPHAMIDE
Instrument ID: U3000
Recording Time: 04/12/15 11:28:22 Sequence: CYCLOPHOSPHAMIDE Table 6: Peak result for standard No.
Ret.Time (min)
Peak Name
1
2.12
n.a.
Total:
Height
(mAu)
98.834
Area (mAU*min)
Rel.Area (%)
1285.156
100.00
1285.156
100.00
Table 7: IR interpretation of standard cyclophosphamide drug Wave number 3400cm-1 1100 cm-1 1985 cm-1 1300 cm-1 950 cm-1 2370cm-1
Functional group N-H stretch(weak) N-H Stretch (medium) P=0 Stretch -CH2-Cl C-N O=P-OH(singular –strong)
Table 8: IR interpretation of standard cyclophosphamide drug and physical mixture Wave number -1
Functional group
1100 cm
N-H Stretch (medium)
2370cm-1
O=P-OH(singular –strong)
1300 cm-1
-CH2-Cl
3100 cm-1, 1790 cm-1
Conjugated alkane
2750 cm-1
OH-C=0
-1
3690cm
O-H(aliphatic)
1780 cm-1
C=O(stretch)
-1
N-H (stretch)
1250 cm
Table 9: Solubility of cyclophosphamide in various solvent Solvent
Solubility
Water
Soluble
Acetic acid
Rapidly soluble
Ethanol
Freely soluble
Acetic anhydride
Soluble
Diethyl ether
Soluble
Carbon tetrachloride
Soluble
Methanol (acetone free )
Rapidly soluble
Dichloromethane
Slightly insoluble (cloudy formation)
Glacial acetic acid
Slightly insoluble
Ethanol
Soluble
9. ACHOWLEDGEMENTS: The authors acknowledged Emcure Pharmaceuticals Ltd. Ahmedabad for gifting the cyclophosphamide drug, Supra drug and food testing and research laboratory-Himatnagar, for HPLC studies. B.Pharmacy college–Rampura & Ganpat University-Mehsana for providing facilities to carry out research work, MS University–metallurgy department for carrying out XRD and SEM analysis, and finally RK University to providing all necessary assistance. 10. CONFLICT OF INTEREST: Authors reports no conflict of interest. 11. COPYRIGHT TRANSFER The authors of this manuscript hereby transfer all the copyrights and publishing rights to RK University (Rajkot, India). As per the copyright transfer agreement, RK University reserves the right to publish, distribute, upload, or advertise the contents of this manuscript as and when required, without any restrictions. The authors further acknowledge that the content presented in this manuscript is entirely original in nature and not published anywhere else.
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