Field of invention
The present invention relates to development of gradient high performance liquid chromatography method for the determination of seven of related substances in oseltamivir phosphate employing ultraviolet detection.
Background of the invention
Oseltamivir;(3/?,4/?,55)-4-acetylamino-5-amino-3-(l-ethylpropoxy)-l-cyclohexene-l-carboxylic acid ethyl ester, belongs to a group of selective and very effective neuraminidase inhibitor drug. It acts as a transition state analogue inhibitor of influenza neuraminidase, preventing new viruses from emerging new infected cells. Oseltamivir is an antiviral drug that is used in the treatment and prophylaxis of both influenzavirus A and influenzavirus B.
Oseltamivir was the first orally active neuraminidase inhibitor commercially developed. It is a prodrug, which is hydrolysed, hepatically to the active metabolite, the free carboxylate of oseltamivir (GS4071). It was developed by Gilead Sciences as disclosed in US 5763483 and is currently marketed by Hoffmann-La Roche (Roche) under the trade name Tamiflu. Oseltamivir phosphate, (3R,4R,5S)-4-acetylamino-5-amino-3-(l-ethylpropoxy)l-cyclohexene-1-carboxylic acid, ethyl ester, phosphate (1:1), is a highly water soluble and non-hygroscopic pro-drug of the active metabolite, oseltamivir carboxylate.
A rapid, specific high performance liquid chromatography method has been developed for quantification of related impurities in Oseltamivir phosphate.
As such, a method for the analysis of these related substances was required. It was desirable to have a single method for oseltamivir and the seven known related substances. Therefore, a method was developed to this end.
Summary of the Invention
The present invention provides a method of detecting Oseltamivir and related substances in Oseltamivir phosphate active pharmaceutical ingredient:
a. applying the Oseltamivir phosphate active pharmaceutical ingredient on reverse-phase high performance liquid chromatography column;

b. eluting the Oseltamivir phosphate active pharmaceutical ingredient with a gradient
mobile phase comprising a volatile buffer and acetonitrile;
c. monitoring effluent from the column with an ultraviolet detector to detect a major peak
corresponding to Oseltamivir.
The present invention provides a method of determining the purity of Oseltamivir phosphate active pharmaceutical ingredient:
a. applying the Oseltamivir phosphate active pharmaceutical ingredient on a reverse-phase
high performance liquid chromatography column;
b. eluting the Oseltamivir phosphate active pharmaceutical ingredient with a gradient
mobile phase comprising a volatile buffer and acetonitrile;
c. monitoring effluent from the column with an ultraviolet detector to detect;
i. a major peak corresponding to the Oseltamivir;
ii. optionally one or more other peaks corresponding to one or more related
substance in the Oseltamivir phosphate active pharmaceutical ingredient; and d. measuring one or more characteristics of all peaks detected by detector to calculate related substance content in Oseltamivir phosphate active pharmaceutical ingredient.
The present invention provides a method of determining the amount of a related substance in a Oseltamivir phosphate active pharmaceutical ingredient:
a. Determining the response factor for the related substances by the method comprising; i) running a known amount of the related substance and a known amount of Oseltamivir on reverse-phase HPLC column eluted with a mobile phase, wherein HPLC is outfitted with a UV detector.
ii) monitoring column effluent with a UV detector to detect a related substance absorption peak at a detection wavelength, the absorption peak corresponding to the related substance;
iii) monitoring column effluent with a UV detector to detect a Oseltamivir absorption peak at a detection wavelength, major absorption peak corresponding to the Oseltamivir; and

iv) calculating the response factor of the related substance using peak areas of
the related substance and Oseltamivir standard absorption peak b. Determining the amount of the related substance in a test sample by the method comprising;
i) running the Oseltamivir phosphate active pharmaceutical ingredient under the same assay conditions of step a) to detect another absorption peak corresponding to the related substance; and
ii) calculating the amount of the related substance in Oseltamivir phosphate active pharmaceutical ingredient using response factor.
The present invention further provides a system for detecting related substance in a Oseltamivir phosphate, comprising:
a) High performance liquid chromatography column comprising
i) Kromasil C18 250*4.6mm 5ja column
ii) a gradient mobile phase (20mM ammonium acetate and acetonitrile as organic phase) with a flow rate of 1.0 ml/min
b) Photodiode array (PDA detector).
Detailed description of the Invention
The present invention provides a HPLC based method of detecting and quantitating related substances in Oseltamivir phosphate employing ultraviolet detection which involves applying the Oseltamivir phosphate on reverse-phase high performance liquid chromatography column eluted with a gradient mobile phase comprising a volatile buffer and acetonitrile. Oseltamivir phosphate active pharmaceutical ingredient used for this invention was prepared as per the process disclosed in US 5886213.
Effluent from the column were monitored with an ultraviolet detector to detect a current peak corresponding to Oseltamivir (I) and /or potentially an related substance present in the oseltamivir phosphate. In some embodiments, the use of a LC-MS facilitates identification of related substance detected in the resulting chromatogram. Identified related substance in a

Oseltamivir Phosphate can be further quantified using given HPLC method with photometric detection (HPLC-UV).

Seven related substances in Oseltamivir Phosphate that can be detected by the methods and systems herein include, for example Isopropyl analog (II), Methyl ester (III), Butyl analog (IV), Dihydro (V), Isomer of Oseltamivir (VI), Propionate analog (VII) and OSL 11 (VIII). In some embodiments, oseltamivir phosphate includes oseltamivir which make up the major component by weight in the oseltamivir phosphate. The oseltamivir phosphate can optionally include other minor amounts of component that can be referred to as related substance. In some embodiments, oseltamivir phosphates are batches of substantially pure oseltamivir prepared by chemical synthetic procedures that often contain small amounts of related substance. "Related substances" refers to component other than oseltamivir that is the subject of study. In some embodiments, one or more related substances can make up less than about 1.0% or less than about 0.3%, by weight of the test sample. A related substances can often be another analog of oseltamivir or degradation products or carry overs from chemical synthesis of the synthesis of oseltamivir. In some embodiments, the related substances include one or more of the Compounds Nos (II-VIII) shown in figure:
Figure Removed

0

Quantitation of Oseltamivir related substances with HPLC-UV
Suitable mobile phase composition for the HPLC-UV assay can be any combination of liquid components that effectively elutes the desired oseltamivir, allows for the separation of oseltamivir from potential related substances, and allows photometric detection of oseltamivir at the detection wavelength.
The mobile phase of the HPLC-UV assay can contain water, organic solvent, or a mixture thereof. Any suitable organic solvent that is miscible with water and does not interfere with detection of the oseltamivir at the detection wavelength can be used. In some embodiments, the organic solvent can be acetonitrile, methanol or tetrahydrofuran. In some embodiments, the organic solvent is acetonitrile.
The mobile phase of the HPLC-UV assay can further include a buffer to stabilize a solution at a desired pH. Any buffer that does not interfere with the detection of the oseltamivir at the detection wavelength can be used. In some embodiments, the buffer comprises an ammonium salt such as ammonium acetate. Buffer concentration can be, for example, 10 to about 30 mM. In some embodiments, buffer concentration is about 15 to about 25 or about 18 to about 23 mM. In further embodiments, the volatile buffer salt is present in the mobile phase at a concentration of about 20 mM. Any pH at which the oseltamivir is sufficiently stable such that it can be detected by the methods and systems of the invention is suitable. In further embodiments, the mobile phase has a pH of about 6 to about 7. In yet further embodiment, mobile phase has a pH of about 6.5.
The mobile phase can be run through the HPLC column as an isocratic elution or gradient elution. In embodiments where a isocratic mobile phase is applied, the solvent composition remains constant throughout the analysis the elution. In embodiments where a gradient mobile

phase is applied, the mobile phase can be comprised of a mixture of two or more different eluant solutions, the proportions of which vary over the time course of the elution. For example, the mobile phase can contain variable amounts of organic solvent and buffer during elution. The variation in component amounts can be adjusted such that the gradient mobile phase maintains substantially constant absorbance at the detection wavelength during the course of elution. The variation in component amounts can also be adjusted to optimize peak shape, elution time, separation of oseltamivir from related substances, and other parameters.
In some embodiment, of the mobile phase the first eluant solution, was initial isocratic elution of 15% acetonitrile, followed by varied elution with one of or a mixture of two eluant solutions, each containing different amounts of organic solvent and buffer. In some emodiments a first eluant contains about 10 to about 30 mM buffer, and a second eluant contains about 100% acetonitrile. In further embodiments, a first eluant contains about 20 mM buffer and a second eluant solution contains 100% acetonitrile. At any point during elution, the mobile phase can be composed of 100% a mixture of the two.
The stationary phase of the HPLC-UV assay can be composed of any reverse phase solid support medium that in combination with the mobile phase allows for the detection of oseltamivir and separation of oseltamivir from the seven related substances. In some embodiments, the stationary phase consists of C8 to CIS matrix. In further embodiments, the stationary phase is a C-18 matrix. In yet further embodiments the stationary phase is Kromasil C 18 column.
Oseltamivir phosphate can be diluted to form limit standard sample for introduction into the column. The limit standard sample can have a oseltamivir concentration of about 0.1 to about Img/ mL. Sample diluent can be comprised of 20 mM ammonium acetate buffer with 100% acetonitrile (in a ratio of 70:30) and having the pH 6.5.
The UV detector monitoring effluent from the column can include any spectrophotometer capable of detecting absorption or transmission of UV wavelengths through a liquid sample. The detector can be tuned to a detection wavelength which can be constant for the duration of elution. In some embodiments, effluent is monitored at a wavelength of about 210-360 nm.
The UV response factor (normalized peak area ratio of related substances to oseltamivir at a detection wavelength) for each related substance identified by LC-MS can be determined by carrying out the above HPLC-UV assay on a reference sample containing a known amount of the

related substances as well as a reference sample containing a known amount of the oseltamivir in
high purity.
For example, response factor for a related substance can be calculated according to the following
equation:
Response factor = (Normalized peak area of related substance)/(Normalized peak area of
Oseltamivir)
Where:
Normalized peak area of related substance = (Peak area of related substance) x (%
purity)/(concentration of related substance)
Normalized peak area of Oseltamivir = (Peak area of oseltamivir) x (% purity)/(concentration of
oseltamivir)
Amount of related substance in a Oselatamivir phosphate containing an unknown amount of related substance can be determined by identifying the related substance using LC-MS described herein, followed by calculation of a response factor for the identified related substance by carrying out the above-described HPLC-UV assay on a reference sample of the identified related substance and reference sample of the oseltamivir, assaying a Oseltamivir phosphate according to the HPLC-UV assay described above and using the calculated response factor to calculate the amount of related substance in the Oseltamivir phosphate.
The invention is described in greater detail by way of examples. The following example are offered for illustrative purpose, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results.
EXPERIMENTAL Example Identification and Quantitation of related substances in Oseltamivir Phosphate
According to the procedure below, Seven related substances were present in batches of oseltamivir phosphate for use as an API. Amounts of detected related substances were also quantited.

TABLE REMOVED
Reference samples of each of the above Seven suspected related substance as well as Oseltamivir phosphate were available from in-house source and their structure were verified by LC-MS.
TABLE REMOVED

Response factors for each of the above related substances were determined by the following procedure. Reference samples of each of the seven related substances were assayed by HPLC using photometric detection according to the HPLC-UV parameters provided below.
Instrumentation
The following equipment operated according the manufacturers instruction was used for
obtaining HPLC chromatograms. Equipments with comparable performance can be
substituted.
Vacuum Degasser: Waters 2695 Alliance HPLC

Pump: Waters 2695 Alliance HPLC Injector: Waters 2695 Alliance HPLC Column: Kromasil CIS, 250 * 4.6mm, 5 um.
Detector: 2996 Photodiode Array Detector (Waters Corporation, Milford, MA) Wavelength= 220 nm Temperature: 25°C. Data System: Waters Empower 2 Data Acquisition (Waters Corporation, Milford, MA).
Working Solutions
Eluent A: 20mM ammonium acetate buffer, filtered through a 0.45 mm membrane filter pH 6.50 ±0.02
Eluent B: 100% acetonitrile (HPLC grade, Merck)
Sample Diluent: Combined Mobile Phase A with Mobile Phase B (70: 30 v/v) and mix. HPLC Analysis
Sample analysis was carried out under the following parameters: Flow rate: 1.0 ml/min Injection volume: 20uL Run time: 45 min Wavelength: 220 nm Sample concentration: Img/mL A gradient mobile phase was applied according to Table 1 below
Table 1.
TABLE REMOVED

The normalized peak area for each related substances reference sample and Oseltamivir reference sample was calculated using Equation 1 and the response factor was calculated using equation 2

Equation 1
Normalized peak area of related substance =
(Peak area of related substance) x (% purity)/(concentration of related substance)
Normalized peak area of Oseltamivir =
(Peak area of oseltamivir) x (% purity)/(concentration of oseltamivir) Equation 2
Response factor = Normalized peak area of related substance/ Normalized peak area of Oselatamivir Calculated response factors for each of the 7 related substances using the following data
TABLE REMOVED

Amounts of each of the related substance in a test sample of Oseltamivir Phosphate was determined by running the test sample on the HPLC-UV column described above and using the response factors in the table above using the following equation 3: Equation 3

% in sample = Sample Area v Standard Dilution Potency of Standard
• A X
100
Standard Area Sample Dilution
Oseltamivir Standard Dilution: 0.01 mg/mL Sample Dilution: Img/mL Potency of Standard Oseltamivir : 94.92 % Area of Standard Oseltamivir : 289102 Results are provided in table below

X 100 X Response Factor

TABLE REMOVED
It is to be understood that various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the claims.

WE CLAIM:
1. A method of detecting Oseltamivir and related substances in Oseltamivir phosphate active
pharmaceutical ingredient:
a. applying the Oseltamivir phosphate active pharmaceutical ingredient on reverse- phase
high performance liquid chromatography column;
b. eluting the Oseltamivir phosphate active pharmaceutical ingredient with a gradient
mobile phase comprising a volatile buffer and acetonitrile;
c. monitoring effluent from the column with an ultraviolet detector to detect a current peak corresponding to Oseltamivir.
2. The method of claim 1 wherein said reverse- phase high performance liquid chromatography
column is Kromasil CIS 250*4.6mm 5u column.
3. The method of claim 1 wherein said volatile buffer is ammonium acetate.
4. The method of claim 1 wherein said mobile phase has a pH of about 6.5.
5. The method of claim 1 wherein said mobile phase comprises a mixture of eluant A and eluant
B, the relative amounts of which vary during the course of elution, wherein eluant A consists
essentially of about 20 mM buffer and eluant B consist 100% acetonitrile.
6. A method of determining the purity of Oseltamivir phosphate active pharmaceutical
ingredient:
a: applying the Oseltamivir phosphate active pharmaceutical ingredient on a reverse-phase
high performance liquid chromatography column;
b: eluting the Oseltamivir phosphate active pharmaceutical ingredient with a gradient mobile
phase comprising a volatile buffer and acetonitrile;
c: monitoring effluent from the column with an ultraviolet detector to detect.
i) a major peak corresponding to the Oseltamivir;
ii) optionally one or more other peaks corresponding to one or more related substance in the Oseltamivir phosphate active pharmaceutical ingredient; and

d. measuring one or more characteristics of all peaks detected by detector to calculate related substance content in Oseltamivir phosphate active pharmaceutical ingredient.
7. A method of determining the amount of related substance in Oseltamivir phosphate active pharmaceutical ingredient:
a. Determining the response factor for the related substances by the method comprising;
i) running a known amount of the related substance and a known amount of
Oseltamivir on reverse-phase HPLC column eluted with a mobile phase, wherein
HPLC is outfitted with a UV detector, ii) monitoring column effluent with a UV detector to detect a related substance
absorption peak at a detection wavelength, the absorption peak corresponding to the
related substance; iii) monitoring column effluent with a UV detector to detect a Oseltamivir absorption
peak at a detection wavelength, major absorption peak corresponding to the
Oseltamivir; and
iv) calculating the response factor of the related substance using peak areas of the related substance and Oseltamivir standard absorption peak
b. Determining the amount of the related substance in a test sample by the method
comprising;
i) running the Oseltamivir phosphate active pharmaceutical ingredient under the same assay conditions of step a) to detect another absorption peak corresponding to the related substance; and
ii) calculating the amount of the related substance in Oseltamivir phosphate active pharmaceutical ingredient using response factor.