A Validated HPLC Method For The Determination Biology Essay

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Nocodazole (NCD) is an anti-neoplastic agent which exerts its effect in cells by interfering with the polymerization of microtubules. As NCD affects the cytoskeleton, it is often used in cell biology experiments as a control: for example, some dominant negative Rho small GTPases cause a similar effect as NCD, and constitutively activated mutants often reverse or negate the effect. In this study, NCD was loaded into Solid Lipid Nanoparticle (SLN) systems. A reverse phase high performance liquid chromatograpy (HPLC) method was validated and applied for the determination of NCD in SLN. Determination and validation studies were carried out on a 4.6 - 150 mm, 5µm C18 Thermo column using an optimized mobile phase (MP) of methanol:water:phosphate buffer (45:42.5:12.5, v/v/v) at a flow rate of 0.8 mL.min-1. Diode array detection was performed at 256 nm and the column temperature was adjusted to 40oC. Naproxen was used as an internal standard (IS). The retention times for naproxen and NCD were 2.2 and 9.8 min, respectively. The specified working range was derived from linearity studies and kept in the concentration range 0.5-100 ppm. Limit of dedection (LOD) and limit of quantitation (LOQ) values were determined to be 0.065 ppm and 0.196 ppm, respectively. NCD recovery % results of the SLN formulations stored at 25oC, 4oC and 40oC were investigated and compared to the freshly prepared samples.

NCD is a classical aneugen, which binds to beta-tubulin with high affinity and affects polymerisation kinetics even at very low concentrations. In addition, chemicals like colchicine or NCD may alter the morphology of centromeres and kinetochores, the sites of attachment for spindle microtubules on the chromosome, and induce malorientation and lagging of chromosomes in mitotic and meiotic cells [Shen et al. 2005; Sackett and Varma 1993; Cimini et al. 2002].

DNA content of reconstructed embryos can be controlled by altering cytoskeletal structures and function using cytoskeletal modifiers such as cytochalasins, demecolcine, or NCD. It has been successfully used for chemically assisted enucleation because it induces condensation of metaphase II chromosomes and membrane protrusion with the condensed chromosomes [Lee at al. 2009; Kawakami at al. 2003].

This study describes sensitive, accurate and precise method for the determination of NCD using HPLC. The method has been validated with respect to precision of peak response, linearity range, specificity and accuracy, LOD and LOQ. The method that validated was used for determination of NCD in the novel SLN formulation.

Method validation was studied and accuracy and reliability were proven. The spesificity of the method was analyzed for SLN systems. It was determined that IS peak and active ingredient did not effected by component in the SLN formulation.

In order to determinate the intra day repeatability of the method, 3 different concentrations of NCD containing samples were prepared. The samples were performed 6 times for each concentration. For inter day repeatability of the method, the same series were prepared on different days. Results obtained from these studies were provided as standart error and relative standard. Intra and inter day repeatability results are given in Table 1.

Measurements performed on three different concentrations (low, medium, high) evaluating the repeatability and reproducibility of the analitical method used seem to verify the precision of the method. Because the coefficient of variation % is below 2%. According to results of the repeatability and reproducibility tests, the method precision was found to be within the targeted intervals. Plesabo SLN formulations were prepared for this study. Any interraption on nocadazole specificity with other components were investigated using the chromatograms obtained.

The chromatograms obtained showed that NCD and IS materials peaks are separated (Fig 2). It was therefore concluded that the method used is spesific.

Table 2: Series prepared for determination of accuracy and % recovery results (n=6)

Table 3: Intra day and interday precision and accuracy results (n=6)

Chromatograms of the calibration set prepared within the NCD were used for the calculation of LOD and LOQ values.

LOD = 3.3 σ / S = (3.3 - 0.003747) / 0.1909 = 0.065 ppm

LOQ = 10 σ / S = (10 - 0.003747) / 0.1909 = 0.196 ppm

(σ= standart deviation of response; S= slope of the calibration curve)

The lowest concentration level used in this method is 0.5 ppm. Because LOD and LOQ values are less than this concentration, it can be concluded that the method used is sensitive.

Methanol:water and Methanol:water:phosphate buffer mixtures at different ratios were tested as the MP. Methanol:water: phosphate buffer (45:42.5:12.5, pH 5.03, 25 mM) were selected as the MP. Because the best retention time, separation and distance of NCD and IS peaks (Fig 2).

After ratios of solvents were determined in the MP, first molarity scanning was performed for phosphate buffer solution which used in the MP. This scanning were performed at 10, 25, 75 and 200 mM. Subsequently, pH scanning was carried out for this solution. This scanning also was performed at pH 3.07, 4.02, 5.03, 6, 6.8 and 7.4. According to width of peak was minimum value, values of theoretical number of layer were maximum values and value of peak asymetry was minumum, 25 mM, pH 5.03 phospahate buffer solution was selected (Figure 3 and 4).

Fig. 4: pH selected of phosphate buffer solution

Flow rate of the MP was tested in the range of 0.8-1.5 mL.min-1. Due to the most appropriate peak shape and retention time, a flow rate was selected 0.8mL.min-1. Appropriate the back pressure was seemed to be 102 bar in this flow rate. Injection volume was kept constant at 20µL for all validation studies. Solvent front was observed almost at 2.2 minutes.

Column volume was calculated by multiplying the solvent front time with the flow rate.

Column volume = 0.8 mL.min-1 - 2.2 min. = 1.8 mL

Due to was used nearly 1% of column volume as the injection volume, the volume was used at 20 µL for all validation studies.

Oven temperature was tested at 25oC and 40oC. It was observed that NCD indicated sharper peak symmetry by the increase in temperature. Although, retention time of NCD was raised, low temperature not showed any effect on Rt of IS. Oven temperature was found at nearly 40oC. All validation studies were carried out at 40oC.

Firstly, due to the apolar structure of the subtance 4.6 - 250 mm, 5µm C18 Thermo column was tested. Subsequently in an afford to decrease the retention times and to ensure the best separation of the IS materials peak and NCD peak, 4.6 - 150 mm, 5µm Thermo column was tested and as a results 4.6 - 150 mm, 5µm Thermo column was used in the study.

In this study, naproxen, diclofenac and paracetamol were tested as IS materials. Naproxen was choosen as an IS because of good resolution between the compounds and suitible retention time of the compouds.

System suitability testing is a integral part of many analitycal precedures. The tests are based on the concept that the equipment, elektronics, analitycal separations and samples to be analyzed constitute an integral system to be evaluated.

Calibration, equipment, model and operation information for system suitability testing are shown in Table 6.

Naproxen and NCD peak morphologies were calculated by the software of the HPLC device using the chomatograms derived from the investigations in Table 7.

A shimadzu HPLC device in AUBIBAM was used fort he HPLC process validation and active ingredient determinations.


3.1. Materials

Compritol, NCD, and polyoxyethylene sorbitan monooleat (Tween 80) were purhased from Merck Schuchardt (Germany) and Acros Organics (USA), respectively. The mobile phase, analytical reagent grade methanol for the high performance liquid chromatography were purchased from Merck KgaA (Germany). Used as IS materials naproxen was provided by Abdi Ibrahim (Turkey).

3.2. Preparation of solid lipid nanoparticles (SLN)

Hot homogenization technique was used to prepare NCD loaded SLN. According to this tecnique, 3% lipids, 5% NCD and 1.2% surface active agent (Tween 80) were used. Lipids were melted about 800C. After melted of the lipids, added NCD at the same temparature. Then, tween 80 was added slowly through ultraturaks 20500 rpm, about 1 min NCD loaded SLN obtained.

HPLC method validation

The analytical process validation method Q2(R1) of the International Harmonization Committee was used in this study and the parameters such as linearity, accuracy, precision, specificity were evaluated (ICH Q2B 1996, ICH Q2(R1) 2005).

The operating conditions applied d using the validation process are given in Table 11.

Naproxen was selected as internal standard (IS). 5 mg accurately weighed naproxen was mixed with the MP and 20mL solution was obtained. Later, this solution was dilued at 1:10 ratio.

5mg NCD was accurately weighted and dissolved in DMSO and volume adjusted to 5 mL. Nine samples of 5µL, 25 µL, 50 µL, 250 µL and 500 µL were taken from this stock solution and diluted to 5 mL with DMSO.

In order to establish of linerity a minimum of 5 different concentrations is recommended. The stock solutions of IS and NCD in MP were used to prepare 5 sample solutions of varying NCD concentration.

In order to determinate of linearity, three different series of the same concentration were preparated. The NCD series are 0.5 (low), 10 (medium) and 100 (high) ppm.

The series obtained were injected into the HPLC. Fort he individual peak normalizations (PN) of IS and NCD were calculated by using the area and the retention times (Rt) of chromatograms.

Fig. 5: a: chromatograms of the MP, b: chromatograms of IS, c: chromatograms of NCD, d: chromatograms of IS and NCD

The prepared series were injected into the HPLC and the individual PN for IS and NCD were calculated using the area of chromatograms obtained and the Rt.

PNıs = Areais / Rtis



The mean PN ratios of the three series versus the NCD concentration values were used to calculate the linearity equation.

Preparation of samples

Experiments were repeated 6 times for each sample. Amount of NCD in the SLN formulations were expressed as % recovery. In addition, for the amount in the formulations standart error (SE), relative standart deviation (RSD) and 95% confidence interval (CI) values were calculated [Kim et all 2005; Morel et all 1998].

Determination of nocodazole in SLN

Aproximately 20 mg of SLN suspension was mixed with DMSO to obtain a 10 mL suspension. Then, this suspension was centrifuged at 4000 rpm for 15 min following 5 min in an ultrasonic bath. 1mL of the transparent portion of the suspension was added to 0.6 mL mobile phase with 0.4 mL IS to obtain 2mL of solution and filtered through a 0.2 µm polypropylene filter. 1 mL of this transparent filtrate was applied to the column to calculate the total NCD in the formulation.

Acknowledgements: The authors thank to Dr. Erol ÅžENER for helping validation of HPLC method.

Cimini D, Fioravanti D, Salmon ED, Degrassi F (2002) Merotelic kinetochore orientation versus chromosome mono-orientation in the origin of lagging chromosomes in human primary cells. J Cell Sci 115: 507-15.

ICH Topic Q2B(1996) Validation of analytical prosedures: Methodology. The Europan Agency fort he evaluation of medicinai products. CPMP/ICH/281/95, Step 4, Consensus Guideline.

ICH harmonised tripartite guideline, Validation of analytical prosedures: Text and methodology Q2(R1), Complementary Guideline on Methodology dated 6 November 1996 incorporated in November 2005.

Kawakami M, Tani T, Yabuuchi A, Kobayashi T, Murakami H, Fujimura T, et al. (2003) Effect of demecolcine and nocodazole on the efficiency of chemically assisted removal of chromosomes and the developmental potential of nuclear transferred porcine oocytes. Cloning Stem Cells 5: 379-87.

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(SLN) made of cacao butter and curdlan. European Journal of Pharmaceutical Sciences 24: 199-205.

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Shen Y, Betzendahl I, Sun F, Tinneberg HR, Ritter UE (2005) Non-invasive method to assess genotoxicity of nocodazole interfering with spindle formation in mammalian oocytes. Reproductive Toxicology 19: 459-471.