ABSTRACT
One of the most important parameters describing the liposomal formulation of hydroquinone is encapsulation efficacy. For the efficacy evaluation of hydroquinone trapped in liposomal structure, there is a need to first separate liposome from the matrix surrounding it
There are various separation techniques; however, in this study, the three techniques of centrifuges with and without washing and dialysis were used
From among the laboratory techniques, an appropriate method is the one that offers responses with a high repeatability
The statistical calculations revealed that encapsulation efficacy with a direct method resulted from a separation via the techniques of dialysis and centrifuge without washing had the highest dispersion with SDs of 6.1 and 8.7, respectively, while the SD value in the technique of centrifuge with washing was 5.2. Through an indirect method, hydroquinone encapsulation efficacy showed the best repeatability with SD values of 2.8 and 2.1 by using the two techniques of centrifuge and centrifuge filtration, respectively
It seems that the treatments leading to the dilution of hydroquinone formulation would result in hydroquinone leakage and a reduction of encapsulation efficacy
It seems that measurement of hydroquinone encapsulation efficacy with an indirect method is a better choice; therefore, a centrifuge technique was utilized to report the mentioned efficacy at a speed of 45000 rcf and duration of 30 min due to having a reasonable price and ease of access?
ABSTRACT
The method has been developed and validated for the determination of hydroquinone in liposomal formulation. The samples were dissolved in methanol and evaluated in 293 nm. The validation parameters such as linearity, accuracy, precision, specificity, limit of detection [LOD] and limit of quantitation [LOQ] were determined. The calibration curve was linear in 1-50 microg/mL range of hydroquinone analyte with a regression coefficient of 0.9998. This study showed that the liposomal hydroquinone composed of phospholipid [7.8%], cholesterol [1.5%], alpha ketopherol [0.17%] and hydroquinone [0.5%] did not absorb wavelength of 293 nm if it diluted 500 times by methanol. The concentration of hydroquinone reached 10 microg/mL after 500 times of dilution. Furthermore, various validation parameters as per ICH Q2B guideline were tested and found accordingly. The recovery percentages of liposomal hydroquinone were found 102 +/- 0.8, 99 +/- 0.2 and 98 +/- 0.4 for 80%, 100% and 120% respectively. The relative standard deviation values of inter and intra-day precisions were <%2. LOD and LOQ were 0.24 and 0.72 microg/mL respectively
Subject(s)
Liposomes , Spectrophotometry, Ultraviolet , Validation Studies as TopicABSTRACT
In mammalian system, spermatozoa are not able to fertilize the oocyte immediately upon ejaculation, thus they undergo a series of biochemical and molecular changes which is termed capacitation. During sperm capacitation, signal transduction pathways are activated which lead to protein tyrosine phosphorylation. Tyrosine phosphorylated proteins have an important role in sperm capacitation such as hyperactive motility, interaction with zona pellucida and acrosome reaction. Evaluation of tyrosine phosphorylation pattern is important for further understanding of molecular mechanisms of fertilization and the etiology of sperm dysfunctions and abnormalities such as teratospermia. The goal of this study is to characterize tyrosine phosphorylation pattern in sperm proteins isolated from normospermic and teratospermic infertile men attending Avicenna Infertility Clinic in Tehran. Semen samples were collected and the spermatozoa were isolated using Percoll gradient centrifugation. Then the C with 5% CO[2] in 3% Bovine Serum spermatozoa were incubated up to 6h at 37 Albumin-supplemented Ham's F-10 for capacitation to take place. The total proteins from spermatozoa were extracted and were subjected to SDS-PAGE before and after capacitation. To evaluate protein tyrosine phosphorylation pattern, western blotting with specific antibody against phosphorylated tyrosines was performed. The results upon western blotting showed: 1] at least six protein bands were detected before capacitation in the spermatozoa from normospermic samples. However, comparable levels of tyrosine phosphorylation was not observed in the spermatozoa from teratospermic samples. 2] The intensity of protein tyrosine phosphorylation appears to have been increased during capacitation in the normospermic relative to the teratospermic group. For the first time, these findings demonstrate and suggest that the differences in the types of proteins and diminished tyrosine phosphorylation efficiency in sperm from teratospermic men may be responsible for their compromised capacitation and low fertilization success rates