To validate the quantitative analysis of phospholipid in liposomal amphotericin B for lyophilized injection by UV-Vis spectrophotometer. Liposomal amphotericin B for lyophilized injection produced by Department of Pharmaceutics, Hanoi University of Pharmacy is analyzed with Hitachi model U-1800 spectrophotometer by measuring the amount of phosphor in phospholipid.
Trang 1QUANTITATIVE DETERMINATION OF PHOSPHOLIPID IN
LIPOSOMAL AMPHOTERICIN B FOR LYOPHILIZED INJECTION BY UV-Vis SPECTROPHOTOMETRIC METHOD
Nguyen Tuan Quang 1 ; Nguyen Thi Kieu Anh 3
Nguyen Thai Son 2 ; Pham Thi Minh Hue 3
SUMMARY
Objectives: To validate the quantitative analysis of phospholipid in liposomal amphotericin B for lyophilized injection by UV-Vis spectrophotometer Materials and methods: Liposomal amphotericin B for lyophilized injection produced by Department of Pharmaceutics, Hanoi University of Pharmacy is analyzed with Hitachi model U-1800 spectrophotometer by measuring the amount of phosphor in phospholipid Results: The method was validated for specificity, compatibility, linearity, propriety, accuracy and the phosphor determined ranges from 45.36 to 68.05 µg/mL As a result, the total amount of phospholipid measured in liposomal amphotericin
B for lyophilized injection was 284.11 ± 4.41 mg/vial Conclusion: The appraised procedure obtained all the requirements and can be used to measure the amount of phospholipid in liposomal amphotericin B for lyophilized injection
* Keywords: Phospholipid; Liposomal amphotericin B; Lyophilized injection; UV-Vis spectrophotometer
INTRODUCTION
Liposome is a form of nano - structured
microcyst Nowadays, scientists have
strong research interest in liposome for
various functions, especially in the
pharmaceutical industry [1] Liposome’s
main components include phospholipids
(PL) and cholesterol Many types of PL
used in liposome preparation can be used
seperately or in combination with others
Types and amount of PL in proportion
to cholesterol plays a very important role
in the sustainability and stability of
composed liposome Therefore, apart
from criteria such as quality and quantity
of active elements, measurement of
particle size, the measurement of liposome products quality is required in PL quality control process
Liposomal amphotericin B (AmB) for lyophilized injection produced by Department
of Pharmaceutics, Hanoi University
of Pharmacy contains distearoyl phosphatidylglycerol (DSPG-C42H83O10P) and hydrogenated phosphatidylcholine (HSPC-C44H88NO8P) The combination of these PLs caused difficulties in measuring the total amount of PL in liposome injection AmB because DSPG and HSPC have similar molecular weights (DSPG was 779.076 g/moL, HSPC was 783.774 g/moL)
1 Vietnam Military Medical University
2 103 Military Hospital
3 Hanoi University of Pharmacy
Corresponding author: Nguyen Tuan Quang (dsquang2000@yahoo.com)
Date received: 25/12/2018
Date accepted: 13/02/2019
Trang 2Based on the composition of the two
types of PL in liposome products and the
principle of coloring reaction between
phosphorus and molybdate, which was
published in some studies [2, 3, 4], we
have conducted experiments and presented
in this article: The results of the process
of measuring the total amount of
spectrophotometric method
MATERIALS AND METHODS
1 Materials and instruments
- Liposomal AmB for lyophilized
injection produced by Department of
Pharmaceutics, Hanoi University of
Pharmacy contains mainly AmB, DSPG,
HSPC, and cholesterol
- Placebo sample contains liposomal
AmB for lyophilized injection sample but
not DSPG and HSPC
- Chemicals: Standardized kali
dihydrophosphat (KH2PO4) (Merck, 99.9%),
ammonium molybdate, ascorbic acid,
percloric acid, sulfuric acid, and other
standardized chemical reagents
- Hitachi U-1800 spectrum analyzer
(Japan), Mettler Toledo analytical balance
(Swiss, precision: 0.1 mg), 800B
centrifuge (China) and other standardized
analytical devices
2 Methods
- Preparation for testing solutions:
+ Ammonium molybdate 1%: Dissolve
1 g of ammonium molybdate and fill up to
100 mL with distilled water
+ Ascorbic acid 2%: Dissolve 2 g of
ascorbic and fill up to 100 mL with
distilled water
+ Mixture B: Ammonium molybdate 1%
and ascorbic acid solution 2% in ratio of 2:3 (v/v) (mix well before use)
+ HClO4 70% saturated with ammonium molybdate: Add ammonium molybdate
to 20 mL of HClO4 70%, stir until being indissoluble, centrifuge and extract clear solution
- Sample preparation:
+ Standardized sample: Put 250.0 mg
of KH2PO4 into a 100 mL volumetric flask, dissolve and fill up with distilled water Suck 2 mL into a 20 mL volumetric flask, fill up with water
+ Testing sample: Put 250.0 mg of the product into a beaker of 100 mL Add
10 mL of solid HNO3, boil gently for evaporation until there’s about 5 mL left Cool off and add 3 mL of HClO4, gently heat until HClO4 white smoke appears Continue to boil until the solution become paler (in about 10 minutes) Cool off and move the solution into a 50 mL cooled vat, fill up the vat with water (cool off the vat and the solution while filling up)
+ Placebo sample: Carry out similarly with test sample but using 0.29 g of the placebo sample
+ Blank sample: Distilled water
- Practical procedure: Suck 200 μL of standardized solutions, testing solutions, placebo solution and distilled water into a
100 mL heat - resistant triangular flask, add exactly 1.4 mL of HClO4 70%
saturated with ammonium molybdate, lightly shake, heat gently for about 1 hour (the vase has milky white dregs), cool off Add exactly 8 mL of mixture B, shake well and gently steam at 50 - 55 degrees Celsius for 1 hour, cool off Move the
Trang 3solution into a 10 mL volumetric flask, fill
up with distilled water and shake well
Filtrate using filtration paper; use the
filtrated solution to conduct UV-Vis
spectrometry
Scan the spectrum at the wavelength
of 800 - 840 nm and measure the
absorbance value at λmax = 820 nm
Calculate the results using the measured
absorbance value
The content of phosphorus/vial was
calculated by the following formula:
At * Mc * HLc * 31 * 50
X = * Mtb (mg P/vial)
Ac * 136.1 * 1000 * Mt
In which:
At, Ac: The absorbance of the sample
and standardized sample; Mc and HLc:
The amount (mg) and the content of
standardized KH2PO4 sample; Mt, Mtb:
The weight and average amount of
sample (g); 31: The molecular weight of P
in KH2PO4 (g); 136.1: The molecular
weight of KH2PO4 (g); 50 and 1,000: The
liquescency of sample and standardized samples
Measure the total content of PL (mg) in
a testing sample vial using HSPC with the formula: X * 783.774 /31
- Measuring process appraisal: For the specificity, compatibility, linearity, propriety, accuracy and interval [5]
RESULTS AND DISSCUSION
1 Specificity
Measure the absorbance of the prepared standardized sample, placebo sample and blank sample The results showed that the spectral chart of blank sample did not show maximum absorbance at 820 nm The spectral chart
of testing and standardized samples showed maximum absorbance at 820 nm The spectral chart of placebo sample showed a response to absorbance at
820 nm, but the response was not greater than 1.0% compared to that of the standardized sample
Table 1: Effect of placebo sample on absorbance of active elements
Absorbance Number
Effect of placebo sample (%)
2 Linearity
Put 250.0 mg of KH2PO4 into a 100 mL of volumetric flask, dissolve and fill up with distilled water (original standardized solution) Suck certain amount of the standardized
solution and dilute using distilled water (as described in table 2) to obtain a range of
titrated solutions with concentration of about 50%, 80%, 100%, 120% and 150%
Trang 4compared to quantitative concentration Suck 200 μL of standardized solutions, testing solution, placebo solution and distilled water into 100 mL of heat-resistant triangular flask, then conduct reaction (as above) The results of the correlation between the absorbance of the standardized sample and phosphorus concentration were presented
in table 2 and figure 1
Table 2: Linearity of the measuring process
quantification
Standardized weight (mg)
Dilution (times)
Phosphorus
Regression equation: y = 0.0136 x + 0.0036 Correlation coefficient (r): 0.9982
Corner coefficient: 0.0136 Intercept coefficient: 0.0036
%Y: 0.4609
Figure 1: Linear correlation between phosphorus concentration and absorbance
Results showed that in the testing concentration ranging from 28.35 to 85.06 μg/mL, there was a strong linear correlation between phosphorus concentration and absorbance with correlation coefficient r ≈ 1 Intercept coeficient Y (at the concentration of 56.70 μg/mL) was 0.4609% (satisfaction < 2%)
Concentration p (µg/ml) Absorbance
Trang 53 Propriety
The appraisal of propriety was conducted by adding a standardized sample to the placebo sample to obtain the corresponding standardized phosphorus solutions of 80%, 100% and 120%, respectively to the quantitative concentration Specifically:
- Preparation of standardized solutions: Put about 0.5 g; 0.63 g and 0.75 g of KH2PO4 into three different 50 mL of volumetric flasks, dissolve and fill up with distilled water
- Put 0.29 g of the placebo sample into a 100 mL beaker Add exactly 1.0 mL each
of the original solution (repeat 3 times for each), mix well and fulfill as above (testing sample preparation)
- Suck 200 μL of standard solutions, self - generated sample solution and distilled water into 100 mL heat-resistant flaks, then conduct the reaction (as above)
Table 3: Results of the appraisal on the propriety of the measuring process
quantification
Added standardized
Revovery
Medium (%) of 9 results (n = 9): 99.45
RSD (%) of 9 results (n = 9): 1.10
By using this method, the recovery level at each concentration level was within the allowance range of 98 - 102%, with the RSD of 9 results was 1.10% (within the limit of < 2%) Therefore, the method satisfied the requirements of propriety
Trang 64 Accuracy
* Repeatability:
Repeatability of the measuring process was determined after 6 times of quantification made on one testing sample at the described conditions
Table 4: Results of the repeatability of measuring process (n = 6)
Standardized sample weight : 249.2 mg; standardized sample absorbance: 0.816
The method had high repeatability with RSD = 1.48% (satisfaction < 2%)
* Intermediary accuracy:
Intermediary accuracy of the measuring process was determined in the same way
as that of repeatability but was conducted on a different date and by different testers
Table 5: Results of the intermediary accuracy of the measuring process
Standardized sample: 249.2 mg
Number
Testing
sample
Testing sample weight (g)
Average quantification (12 testing samples): 99.59%
RSD (%) (12 testing samples): 1.45 (%)
Trang 75 Definite interval
Measuring the absorbance for 6 times of 80% and 120% standardized solutions compared to the quantitative concentration (in linearity)
Table 6: Results of the accuracy of the measuring process
Number
Concentration %
compared to quantitative
concentration
Absorbance Number
Concentration % compared to quantitative concentration
Absorbance
The results showed that at both concentration levels of 80% and 120% compared to the quantitative concentrations gave an RSD (%) of < 2% The phosphorus definite interval was from 45.36 to 68.05 μg/mL
6 The total measured amount of PL
in AmB liposome lyophilized injection
From the verified measuring process,
we measured the total content of PL in
AmB liposome lyophilized injection The
results showed that the total PL content in
AmB liposome lyophilized injection was
284.11 ± 4.41 mg/vial (n = 6)
CONCLUSIONS
The process of measuring the PL
proportion in liposomal AmB for lyophilized
injection was appraised by UV-Vis
spectrophotometric method using phosphorus amount measurement The process was evaluated to ensure specificity, compatibility, linearity, propriety, accuracy as required and the defined phosphorous range was from 45.36 to 68.05 µg/mL As a result, the determined proportion of PL in AmB liposome lyophilized injection was 284.11 ± 4.41 mg/vial
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1 Vo Xuan Minh, Pham Thi Minh Hue
Nanotechnology and liposome application
in pharmaceuticals, cosmetics Information
Trang 8Center - Library Hanoi University of Pharmacy
2013, pp.55-59, 84-100
2 Samjhana Pradhan, Megh Raj Pokhrel
Spectrophotometric determination of phosphorus
in sugarcane juice, fertilizer, detergent and
water samples by molybdenum blue method
Scientific World 2013, 11 (11), pp.58-62
3 Sanjeevan J Kharat, Sanjay D Pagay
Determination of phosphate in water samples
of Nashik District (Maharashtra State, India)
river by UV-Visible spectroscopy E-Journal of Chemistry 2009, 6 (S1), pp.515-521
4 Xiao-Lan Huang, Jia-Zhong Zhang
Kinetic spectrophotometric determination of submicron orthophosphate by molybdate reduction Microchemical Journal 2008, 89, pp.58-71
5 Asean Guidelines for validation of
analytical procedures Adopted from ICH guidelines, ICH Q2A (1994), ICH Q2B (1996)