Optimization and validation of an RP-HPLC method for the estimation of 6-mercaptopurine in bulk and pharmaceutical formulations

A reverse phase HPLC method is described for the determination of 6-mercaptopurine in bulk and tablets. Chromatography was carried on a C18 column using a mixture of acetonitrile and 0.05 mol/L sodium acetate buffer (10:90 v/v) as the mobile phase at a flow rate of 1 mL/min-1 with detection at 324 nm. The retention time of the drug was 3.25 min. The detector response was linear in the concentration of 0.01-5 μg/mL. The limit of detection and limit of quantification were 17 and 52 ng/mL respectively. The method was validated by determining its sensitivity, linearity, accuracy and precision. The proposed method is simple, economical, fast, accurate and precise and hence can be applied for routine quality control of mercaptopurine in bulk and tablets.


INTRODUCTION
6-Mercaptopurine (Figure 1) is an anti-cancer ("antineoplastic" or "cytotoxic") chemotherapy drug.In Cancerous tumors, cell division is no longer controlled as it is in normal tissue.When "Normal" cells come into contact with like cells, they stop dividing.This mechanism is called contact inhibition.Cancerous cells do not have this ability.Chemotherapy kills cancer cells by halting cell division.Chemotherapy has been found to be most effective at killing cells that are rapidly dividing.But, since chemotherapy cannot distinguish between the cancerous cells and the normal cells, both are affected.Hence side effects occur until the "normal" cells grow back and be healthy.
6-Mercaptopurine belongs to the class of chemotherapy drugs called "antimetabolites".When antimetabolites are incorporated into cellular metabolism V. Somasekhar

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by cells, they are unable to divide.Antimetabolites attack cells at very specific phases in the cycle.6-Mercaptopurine is converted to 6-thioinosinic acid, which acts as an antimetabolite to inhibit synthesis of adenine and guanine and also to prevent conversion of purine bases into nucleotides.Some 6-mercaptopurine is also converted to thioguanine, which is incorporated into both DNA and RNA to generate defective nucleic acids.Thus the synthesis of nucleic acid and its functions are impaired in several ways and cell mitosis is inhibited.The purine antagonist 6-mercaptopurine (6-MP) has been used as a chemotherapy drug and is usually given orally.When higher doses are required, the drug may be given intravenously.Patients suffering from acute lymphocytic or myelocytic leukemia, lymphoblastic leukemia (especially in childhood cases) and acute myelogenous and myelomonocyticleukemias are often given 6-MP in combination with other chemotherapy drugs.In addition to its role in cancer treatment, 6-MP is used to treat patients suffering from a variety of inflammatory bowel diseases.The drug is also used to prevent rejection following organ transplants.Since 6-MP is widely used in chemotherapy, it is important to develop and validate analytical methods for its determination in pharmaceutical dosage forms.Review of literature has revealed that all methods reported for the estimation of 6-MP are useful in estimating 6-MP in biological samples (Sorouraddin et al., 2011;Hawwa et al., 2009;Umrethia et al., 2006;Oliveira et al., 2004;Su et al., 1999;Mawatari et al., 1998;Sahnoun et al., 1990;Erdmann, Chan, Canafax, 1988;Rudy et al., 1988;Migulla, Prumke, Huller, 1987).So far no systematic HPLC method has been reported for determination of 6-MP in pharmaceutical formulations.This paper reports a rapid and sensitive HPLC method with UV detection, useful for routine quality control of 6-MP in pharmaceutical formulations.The method was validated by parameters such as linearity, accuracy, precision and robustness.

Apparatus
The HPLC used consisted of Hitachi chromatographic system equipped with a Hitachi pump L-7110, Rheodyne universal injector 7725 and Hitachi L-7400 UV-visible detector.The chromatographic studies were performed using Phenomenex ® C 18 , 5 μm, 250 mm x 4.6 mm i.d.column, at ambient temperature.Peak area integration was performed using Winchrom software.A Shimadzu model 1700 double beam UV-visible spectrophotometer with a pair of 10 mm matched quartz cells was employed for determination of absorption maximum.

Reagents and chemicals
HPLC grade acetonitrile and glacial acetic acid were obtained from Rankem (Mumbai, India) and sodium acetate (A.R. grade) was obtained from Sd Fine Chemicals, Mumbai, India.Pure sample of drug was obtained from Sigma Aldrich, India.Ultra-pure water obtained from Milli-Q academic system (Millipore Pvt. Ltd., Bangalore, India) was used to prepare all solutions for the method.

Chromatographic conditions
The process was carried out on C 18 column (5 μm, 250 x 4.6 mm, i.d) using the mobile phase consisting of acetonitrile and 0.05 M sodium acetate buffer in the ratio 10:90 with pH adjusted to 6.8 using HPLC grade glacial acetic acid at a flow rate of 1 mL/min.Wavelength was fixed at 324 nm.The mobile phase was filtered through 0.45 μm membrane filter and degassed.

Preparation of solutions
Stock standard solution of the pure drug was prepared by dissolving 100 mg of 6-MP in 100 mL volumetric flask using 0.1 mol/L sodium hydroxide.Then the volume was made up to the mark with the same solvent to give a final concentration of 1000 μg/mL.Nine standard solutions of 6-MP in the range of 0.01-5.0μg/mL (0.01, 0.05, 0.1, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0 μg/mL) were prepared by subsequent dilution with mobile phase.

Validation
Three series of standard solutions in the range of 0.01-5.0μg/mL were prepared and analyzed as described above.Calibration curves were constructed using three series of standard 6-MP solutions in the range of 0.01-5.0μg/mL.Peak area was recorded for all the peaks and a calibration graph was obtained by plotting peak area versus concentration of 6-MP.To establish the accuracy and intra-day and inter-day precision of the method, six replicate solutions at three different concentrations (0.5, 1.0, 2.5 μg/mL) were assayed on single day and three separate days.

Assay method
Twenty tablets were weighed, crushed and an amount of powder equivalent to100 mg of 6-MP was accurately weighed, transferred to a 100 mL volumetric flask, made up to volume with 0.1 mol/L sodium hydroxide and placed in an ultrasonic bath for 20 min.After filtration through a 0.45 μm membrane filter, the solution was suitably diluted with mobile phase to obtain the required concentration.
20 μL of solution was injected into the HPLC system to obtain the chromatograms for the standard drug solution and the sample solution.
A steady baseline was recorded with the optimized chromatographic conditions.The standard solution of 6-MP was injected and the chromatogram recorded.The retention time of 6-MP was found to be 3.25 min.The sample solution prepared from the tablets was then injected and the amount of drug present was calculated from the calibration curve.

Chromatographic conditions
Various compositions of mobile phase consisting of acetonitrile and sodium acetate (50:50 to 10:90) were used in the study and the composition of 10:90 was selected as it gave best elution, reasonable retention time and least tailing.A pH 6.8 was found to give the best elution, while increased tailing was observed at lower pH values.Flow rates between 0.5 and 1.5 were studied and a flow rate of 1 ml/min was found to give a good separation time and peak shape.
The typical chromatogram obtained for 6-MP is presented in Figure 2.

Linearity
Calibration curves were constructed using three series of standard 6-MP solutions in the range of   I.The linearity of the calibration curve was validated by the high value of the correlation coefficient (r=0.9997).

Limit of detection (LOD) and limit of quantification (LOQ)
The limit of detection and the limit of quantification are defined as LOD=3.3σ/s and LOQ=10 σ/s respectively, where σ denotes standard deviation of y-intercepts of regression lines and s denotes slope of the corresponding calibration curve (ICH 2005).
The limit of detection was determined as 17 ng/mL.The limit of quantification was determined as 52 ng/mL.

Precision
The assay was investigated with respect to system suitability test, method precision and intermediate precision.The system suitability test and method precision were carried out to monitor repeatability and reproducibility.
In order to measure repeatability of the system (system suitability test), five consecutive injections were made and the results were evaluated by considering peak area values of 6-MP.The precision values with their R.S.D. are shown in Table II.The results in Table II indicate that the R.S.D.(%) is less than 2%.
Three different concentrations of 6-MP were analyzed in three independent series in the same day (intra-day precision) and three consecutive days (interday precision), within each series every sample was injected six times.The R.S.D. values of intra-and inter-day studies (

TABLE I -
Statistical data of calibration curves of mercaptopurine Table II) varied from 0.16 to 0.79% showing that the intermediate precision of the method was satisfactory.R.S.D.(%) : relative standard deviation; b bias(%) : [(found -taken)/taken] x 100 a

TABLE III -
Recovery Data for the Proposed RP-HPLC method (n=6)