CLIAMS:We Claim,

1. A capillary electrophoresis method is quantifies Phosphate and Phosphite in Bisphosphonates.

2. A capillary electrophoresis method as per claim1 detects Phosphate and Phosphite peaks using indirect method.

3. A capillary electrophoresis method as per claim1 detects Phosphate and Phosphite peaks using 1,2,4- benzene Tricarboxylic acid buffer as background.

4. A capillary electrophoresis method as per claim1 detects the phosphate peak of relative migration time at 1.2 with respect to Phosphite peak.

5. A capillary electrophoresis method as per claim1 quantifies Phosphate and Phosphite peaks at the level of 0.032% and 0.083% respectively.

6. A capillary electrophoresis method as per claim1 detects Phosphate and Phosphite peaks at the level of 0.011 % and 0.028 % respectively.

,TagSPECI:Field of the invention
The present invention relates to an improved Capillary electrophoresis method for the quantitative determination of phosphate and phosphite in Bisphosphonate.

Background of the invention
Alendronate sodium is chemically known as sodium [4-amino-1-hydroxy-1-(hydroxy-oxido-phosphoryl)- butyl]phosphonic acid trihydrate.
Alendronate sodium is a bisphosphonate drug used for osteoporosis and several other bone diseases. It is marketed alone as well as in combination with vitamin D.
Structure of Alendronate sodium

Bisphosphonates were developed in the 19th century, but were first investigated in 1960’s for the use in disorder of bone metabolism. They are a class of compounds which share a common P-C-P backbone in their structure. The initial rationale for the use in humans was their potential in preventing the dissolution of hydroxyl apatite, the principle bone mineral, and hence arresting the bone loss. Bisphosphonates are used clinically for the treatment of Osteoporosis, Osteitis deformans (Paget’s disease of the bone), bone metastasis (with or without hypercalcemia), multiple myeloma and other conditions that feature bone fragility. In Osteophoresis and Paget’s disease, Alendronate and Residronate are the most popular first-line drugs. High potency intravenous bisphosphonates have shown to modify progression of skeletal metastasis in several forms of cancer, especially breast cancer. Phosphates and Phosphites are the important compounds with biological and chemical significance. These ions are also chemically interested in that they form oligomeric species that means they exist in several oxidation states and can undergo multiple protonation and metal ligand reactions.

Generally, impurities are identified spectroscopically and by other physical methods and then the impurities are associated with a peak position in a chromatogram (or a spot on a TLC plate). Thereafter, the impurity can be identified by its position in the CE chromatogram, which is conventionally measured in minutes, the time required for the analyte to move through the effective length of the capillary, that is, from the sample introduction point to the detection point known as “migration time”.

The objective of this work was to quantitatively determine the content of residual phosphate and phosphite impurities present in Bisphosphonates. Since most of the synthetic routes for preparation of Bisphosphonates were involved from alkyl phosphates and phosphites, the residual phosphate and phosphite were considered to be the potential impurities of Bisphosphonates. During method optimization, all chromatographic parameters were found to enhance precision, accuracy, limit of detection and limit of quantitation of phosphate and phosphite ion peaks, with appropriate analysis run time.

Summary of the invention
In one aspect, the present invention provides a Capillary electrophoresis (CE) method for the quantitative determination of Phosphate and Phosphite in Bisphosphonates.

In one aspect, the CE method described in the present invention has the following advantages when compared with conventional HPLC method using RI detector as per European Pharmacopoeia 6.0 page-2884-2885 methods for determining phosphate and phosphite content in alendronate sodium:
i) Shorter run time only 15.0min whereas in HPLC 35.0min;
ii) Area response is very high in comparison of HPLC method;
iii) Comparatively good separation in comparison of HPLC method; and
iv) Very high S/N ratio compare to HPLC method.

Brief description of drawings
Fig. 1 illustrates the chromatogram of spiked (Phosphate and Phosphite in Alendronate Sodium) sample.

Detailed description of the invention
As used herein, “limit of detection (LOD)” refers to the lowest concentration of analyte that can be clearly detected above the base line signal, is estimated is three times the signal to noise ratio.

As used herein, “limit of quantization (LOQ)” refers to the lowest concentration of analyte that can be quantified with suitable precision and accuracy, is estimated as ten times the signal to noise ratio.
The Phosphate and Phosphite are detected and retention time of peak due to Phosphate and phosphite is about 4.0min and 5.0mn respectively.

According to one aspect of the present invention, there is provided a Capillary Electrophoresis (CE) method for quantifying, by area percent, the amounts of Phosphate and Phosphite present in a sample of bisphosphonates.

According to another aspect of the present invention, there is provided an accurate and well-defined CE method for the determination of Phosphate and Phosphite in the bisphosphonate.

Preferably, the method for determining the amount of Phosphate and phosphite in Alendronate sodium sample comprises the steps of:
a) combining a alendronate sodium sample with water to obtain a solution;
b) injecting the sample solution into column effective length 50cm, Internal diameter 50µm, Outer diameter 375µm;
c) Measuring of the amounts of Phosphate and Phosphite at 217 nm wavelength with a UV detector (having an appropriate recording device).
d) Rinse Pressure is at 30.0psi for 5.0 minutes and inject pressure is 0.5psi for 20seconds.
e) The separate voltage is kept at 15.0KV ad the run time is 10minutes.
f) Buffer preparation: 183.9 m of 1,2,4-Tricarboxylic acid in 60 ml water and adjust pH 3.4 with diluted sodium hydroxide solution then transfer the solution in 100mL volumetric flask and make up the volume with water. Again check the pH and adjust pH 3.5 with diluted sodium Hydroxide Solution with ortho-phosphoric acid solution. Filter it through 0.22µ membrane filter.

The LOD /LOQ values of Phosphate and Phosphites are summarized in Table 1.

Table 1

S. No Components LOD (%) LOQ (%)
1 Phosphate 0.011 0.032
2 Phosphite 0.028 0.083

Preferably, the limit of detection (LOD) and limit of quantification (LOQ) were estimated by signal to noise ratio method, by injecting a diluted solution with known concentration.
The accuracy of the method with the spiked impurities was evaluated at 0.5 % concentration level,
According to another aspect of the present invention, there is provided a capillary electrophoresis method to get the separation of Phosphate and Phosphite from analyte peak. Satisfactory separation was achieved using the buffer consists of (183.9 m of 1,2,4-Tricarboxylic acid in 60 ml water and adjust pH 3.4 with diluted sodium hydroxide solution then transfer the solution in 100mL volumetric flask and make up the volume with water. Again check the pH and adjust pH 3.5 with diluted sodium Hydroxide Solution with ortho-phosphoric acid solution. Filter it through 0.22µ membrane filter.) In the optimized conditions the Phosphate and phosphite were well separated and the typical retention times (RT) of Phosphate and Phosphite were about 5.15 and 4.00 minutes respectively, and typically shown in Figure 1.
The system suitability values of Phosphate and Phosphite were summarized in Table 2.
Table 2
Compound (n=1) Rt Rs N T
Phosphate 5.15 13.10 53163 1.18
Phosphite 4.00 33393 0.69

*n=1: determination, Rt: Migration time, Rs: USP resolution, N: number of theoretical plates (USP tangent method), T: USP tailing factor, m/z: mass number.

The percentage recovery of Phosphate and Phosphite in bulk drug samples was done at 0.5 %. The percentage recovery of Phosphate and Phosphite in bulk drugs samples was ranged from 90.00 to 110.00.

In deliberate varied chromatographic conditions , the robustness of the method is confirmed.

Experimental
The CE system used for method development and was P/ACE MDQ Capillary electrophoresis system (manufactured by Backman coulter) CE system with a photo diode array detector. The out put signal was monitored and processed using 32 Karat software (designed by Backman coulter) on DELL computer (Digital Equipment Co).
The column used was a effective length 50cm, Internal diameter 50µm, Outer diameter 375µm; The buffer (183.9 m of 1,2,4-Tricarboxylic acid in 60 ml water and adjust pH 3.4 with diluted sodium hydroxide solution then transfer the solution in 100mL volumetric flask and make up the volume with water. Again check the pH and adjust pH 3.5 with diluted sodium Hydroxide Solution with ortho-phosphoric acid solution. Filter it through 0.22µ membrane filter.), The Rinse pressure was 30.0psi for 5minutes, The Inject pressure was 0.5psi for 20 seconds and the separate voltage was 15.0KV. The run time was 10 min and the wavelength was monitored at a wavelength of 217 nm. Water was used as diluent during the standard and test samples preparation.

Preparation of Phosphoric acid Stock solution:
3.0 g of phosphoric acid was accurately weighed and transferred to the 100mL volumetric flask (BOROSIL-Class-A), 20 mL of diluent was added in to the flask and shaken for five minutes in an ultrasonic bath and made up to mark with diluent. Dilute 1.0mL of the solution to 100.0mL with water.

Preparation of Phosphorous acid stock solution:
2.5 g of phosphoric acid was accurately weighed and transferred to the 100mL volumetric flask(BOROSIL-Class-A), 20 mL of diluent was added in to the flask and shaken for five minutes in an ultrasonic bath and made up to mark with diluent. Dilute 1.0mL of the solution to 100.0mL with water.

Phosphate-Phosphite Standard solution:
Add 2.0 mL each of Phosphoric acid Stock solution and Phosphorous acid stock solution stock solution into 50 mL volumetric flask and make up the volume with diluent and mix well.