DESC:FIELD OF THE INVENTION

The present invention relates to a bioanalytical method for assay of Betahistine in human plasma. Further present invention discloses process for the bioanalytical method.

BACKGROUND OF THE INVENTION

Betahistine is the International Nonproprietary Name (INN) of N-methyl-2-(pyridin-2-yl)ethanamine having the CAS number 5638-76-6. The structure of Betahistine corresponds to the formula (I)

(I)

Betahistine is a vasodilator agent which is commonly used as its dihydrochloride salt. After oral administration, Betahistine dihydrochloride is almost completely absorbed from all parts of the gastro-intestinal tract; and after absorption, Betahistine dihydrochloride is rapidly and almost completely metabolized into its metabolite 2-pyridylacetic acid (2-PAA).

The first marketing authorization in Europe for Betahistine dihydrochloride was in 1970 for balancing or alleviating vertigo, tinnitus, hearing loss and nausea symptoms associated with Ménière’s disease. The treatment of Ménière’s disease is a long-term treatment which encompasses a first initial oral treatment phase followed by a second maintained phase. Betahistine dihydrochloride is commercially available in form of immediate release tablets of 8 mg, 16 mg or 24 mg or in form of oral solution of 8 mg/ml.

Betahistine chemically is N-methyl-2-(pyridin-2-yl)ethanamine and is commonly used as its dihydrochloride salt. Betahistine is rapidly converted by monoamino oxidase (MAO) enzymes to its major metabolite 2 pyridyl acetic acid (2-PAA), so Betahistine cannot be quantified in human plasma; hence, currently 2-PAA is used as surrogate pharmacokinetics (PK) parameter for measurements for Betahistine PK. Various prior-art literature are available which are related to bioanalytical method for assay of Betahistine.

A presentation by A. Muntendam in EBF Symposium 16th November, 2012 which is available at http://www.e-b-f.eu/wp-content/uploads/2018/05/bcn2012-S52.-4_muntendam.pdf (accessed on 22nd August, 2019) discloses metabolism of Betahistine into 2-PAA can be stopped by cooling and/or adding a MAO inhibitor. Specifically, the presentation discloses use of selegiline as a MAO inhibitor and double liquid-liquid extraction (LLE) method. The presentation is silent about analytical method process and critical process parameters as well critical reagents.

A thesis discloses method development and validation of Betahistine in human plasma by using LC-MS/MS which is available at https://shodhganga.inflibnet.ac.in/bitstream/10603/212232/11/11_chapter%204.pdf (accessed on 22nd August, 2019). The thesis discloses extraction technique for the bioanalytical method is liquid-liquid extraction (LLE).

Murilo B. et al, J. Braz. Chem. Soc., Vol. 30, No. 7, 1415-1424, 2019, discloses characterization and in silico mutagenic assessment of a new Betahistine degradation impurity. Murilo B. et al, is not related to bioanalytical method.

A simple, accurate and precise method for bioanalytical method for assay of Betahistine in human plasma is desired. Particularly bioanalytical method for assay of Betahistine is desired where in Betahistine itself is measured in place of current practice of measuring its metabolite 2-PAA.

Therefore, the present invention provides a simple, accurate and precise method for bioanalytical method for assay of Betahistine in human plasma which measures amount of Betahistine in human plasma.

OBJECT OF THE INVENTION

Object of the present invention is to provide a simple, accurate and precise bioanalytical method for assay of Betahistine in human plasma.

Yet another object of the present invention is to provide a simple, accurate and precise bioanalytical method for assay of Betahistine in human plasma, wherein the method comprises measurement of Betahistine in human plasma without any surrogate pharmacokinetics (PK) parameter.

Yet another object of the present invention is to provide a simple, accurate and precise bioanalytical method for assay of Betahistine in human plasma, wherein the method comprises; (a) measurement of Betahistine in human plasma without any surrogate pharmacokinetics (PK) parameter, and (b) use of ion pairing reagent in mobile phase.

Yet another object of the present invention is to provide a simple, accurate and precise bioanalytical method for assay of Betahistine in human plasma, wherein the method comprises; (a) measurement of Betahistine in human plasma without any surrogate pharmacokinetics (PK) parameter, and (b) use of Heptafluorobutyric acid (HFBA) as ion pairing reagent in mobile phase.

Yet another object of the present invention is to provide a simple, accurate and precise bioanalytical method for assay of Betahistine in human plasma, wherein the method comprises; (a) measurement of Betahistine in human plasma without any surrogate pharmacokinetics (PK) parameter, (b) use of Heptafluorobutyric acid (HFBA) as ion pairing reagent in mobile phase, and (c) use of single solid phase extraction (SPE) method for extraction of Betahistine.

Yet another object of the present invention is to provide a simple, accurate and precise bioanalytical method for assay of Betahistine in human plasma, wherein the method comprises; (a) mixing selegiline and human plasma sample comprising Betahistine, (b) use of single solid phase extraction method for extraction of Betahistine, and (c) using Heptafluorobutyric acid as ion pairing reagent in mobile phase and in reconstitution solution in the assay of Betahistine in human plasma using LC-MS/MS, wherein the method measures Betahistine itself in human plasma without any surrogate pharmacokinetics parameter.

Yet another object of the present invention is to provide a simple, accurate and precise bioanalytical method for assay of Betahistine in human plasma, wherein the method comprises step of mixing monoamino oxidase inhibitor (MAOI) and human plasma sample comprising Betahistine.

Yet another object of the present invention is to provide a simple, accurate and precise bioanalytical method for assay of Betahistine in human plasma, wherein the method comprises: (a) mixing monoamino oxidase inhibitor (MAOI) and human plasma sample comprising Betahistine, (b) use of Heptafluorobutyric acid (HFBA) as ion pairing reagent in mobile phase, and (c) use of single solid phase extraction (SPE) method for extraction of Betahistine, wherein, the method measures Betahistine itself.

Yet another object of the present invention is to provide a simple, accurate and precise bioanalytical method for assay of Betahistine in human plasma, wherein the method comprises measurement of Betahistine in human plasma without any surrogate pharmacokinetics (PK) parameter, wherein the method comprises: (a) mixing selegiline and human plasma sample comprising Betahistine, (b) use of Heptafluorobutyric acid (HFBA) as ion pairing reagent in mobile phase, and (c) use of single solid phase extraction (SPE) method for extraction of Betahistine.

SUMMARY OF THE INVENTION

The present invention provides a simple, accurate and precise bioanalytical method for assay of Betahistine in human plasma, wherein the method comprises measurement of Betahistine in human plasma without any surrogate pharmacokinetics parameter, wherein the method comprises mixing monoamino oxidase inhibitor and human plasma sample comprising Betahistine. Further, the method uses heptafluorobutyric acid as ion pairing reagent in mobile phase, and single solid phase extraction method for extraction of Betahistine.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a simple, accurate and precise bioanalytical method for assay of Betahistine in human plasma.

In another embodiment, the present invention provides a simple, accurate and precise bioanalytical method for assay of Betahistine in human plasma, wherein the method comprises measurement of Betahistine in human plasma without any surrogate pharmacokinetics (PK) parameter.

In another embodiment, the present invention provides a simple, accurate and precise bioanalytical method for assay of Betahistine in human plasma, wherein the method comprises: (a) measurement of Betahistine is in human plasma without any surrogate pharmacokinetics (PK) parameter, and (b) use of ion pairing reagent in mobile phase.

In another embodiment, the present invention provides a simple, accurate and precise bioanalytical method for assay of Betahistine in human plasma, wherein the method comprises: (a) measurement of Betahistine in human plasma without any surrogate pharmacokinetics (PK) parameter, and (b) use of Heptafluorobutyric acid (HFBA) as ion pairing reagent in mobile phase.

In another embodiment, the present invention provides a simple, accurate and precise bioanalytical method for assay of Betahistine in human plasma, wherein the method comprises: (a) measurement of Betahistine in human plasma without any surrogate pharmacokinetics (PK) parameter, and (b) use of Heptafluorobutyric acid (HFBA) as ion pairing reagent in mobile phase B and in reconstitution solution.

In another embodiment, the present invention provides a simple, accurate and precise bioanalytical method for assay of Betahistine in human plasma, wherein the method comprises: (a) measurement of Betahistine in human plasma without any surrogate pharmacokinetics (PK) parameter, (b) use of Heptafluorobutyric acid (HFBA) as ion pairing reagent in mobile phase, and (c) use of single solid phase extraction (SPE) method for extraction of Betahistine.

In another embodiment, the present invention provides a simple, accurate and precise bioanalytical method for assay of Betahistine in human plasma, wherein the method comprises: (a) measurement of Betahistine in human plasma without any surrogate pharmacokinetics (PK) parameter, (b) use of Heptafluorobutyric acid (HFBA) as ion pairing reagent in mobile phase B and in reconstitution solution, and (c) use of single solid phase extraction (SPE) method for extraction of Betahistine.

In another embodiment, the present invention provides a simple, accurate and precise bioanalytical method for assay of Betahistine in human plasma, wherein the method comprises; (a) mixing selegiline and human plasma sample comprising Betahistine, (b) use of single solid phase extraction method for extraction of Betahistine, and (c) using Heptafluorobutyric acid as ion pairing reagent in mobile phase and in reconstitution solution in the assay of Betahistine in human plasma using LC-MS/MS, wherein the method measures Betahistine itself in human plasma without any surrogate pharmacokinetics parameter.

In another embodiment, the present invention provides a simple, accurate and precise bioanalytical method for assay of Betahistine in human plasma, wherein the method comprises step of mixing monoamino oxidase inhibitor (MAOI) and human plasma sample comprising Betahistine.

In another embodiment, the present invention provides a simple, accurate and precise bioanalytical method for assay of Betahistine in human plasma, wherein the method comprises: (a) Mixing monoamino oxidase inhibitor (MAOI) and human plasma sample comprising Betahistine, (b) Use of Heptafluorobutyric acid (HFBA) as ion pairing reagent in mobile phase, and (c) Use of single solid phase extraction (SPE) method for extraction of Betahistine, wherein, the method measures Betahistine itself.

In another embodiment, the present invention provides a simple, accurate and precise bioanalytical method for assay of Betahistine in human plasma, wherein the method comprises: (a) Mixing selegiline and human plasma sample comprising Betahistine, (b) Use of Heptafluorobutyric acid (HFBA) as ion pairing reagent in mobile phase B and in reconstitution solution, and (c) Use of single solid phase extraction (SPE) method for extraction of Betahistine, wherein, the method measures Betahistine itself.

In another embodiment, the present invention provides a simple, accurate and precise bioanalytical method for assay of Betahistine in human plasma, wherein the method comprises measurement of Betahistine in human plasma without any surrogate pharmacokinetics (PK) parameter, wherein the method comprises: (a) mixing selegiline and human plasma sample comprising Betahistine, (b) use of Heptafluorobutyric acid (HFBA) as ion pairing reagent in mobile phase, and (c) use of single solid phase extraction (SPE) method for extraction of Betahistine.

In another embodiment, the present invention provides a simple, accurate and precise bioanalytical method for assay of Betahistine in human plasma, wherein the method comprises measurement of Betahistine in human plasma without any surrogate pharmacokinetics (PK) parameter, wherein the method comprises: (a) mixing selegiline and human plasma sample comprising Betahistine, (b) use of Heptafluorobutyric acid (HFBA) as ion pairing reagent in mobile phase B and in reconstitution solution, and (c) use of single solid phase extraction (SPE) method for extraction of Betahistine.

The inventor of the present invention have developed and validated a novel and highly reproducible bioanalytical method to quantify concentration of Betahistine.

The bioanalytical method for assay of Betahistine of the invention is used for quantification of Betahistine in human plasma after administering Betahistine composition to the patient in need thereof. The composition for administration to patient in need thereof comprises Betahistine or a pharmaceutically acceptable salt thereof. The term “pharmaceutically acceptable salt” used herein encompasses a salt formed from pharmaceutically acceptable non-toxic acids including inorganic or organic acids. There is no limitation regarding the salts, except that if used for therapeutic purposes, they must be pharmaceutically acceptable. Salts of betahistine can be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include among others acetic, benzene sulfonic, benzoic, camphor sulfonic, citric, ethansulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, lactic, maleic, malic, mandelic, methanesulfonic, phosphoric, succinic, sulphuric, tartaric, and p-toluensulfonic acid.

Specifically, the composition for administration to the patient in need thereof is the composition disclosed in patent application WO2019016668 which is incorporated herein as reference. The composition for administration to the patient in need thereof is prolonged-release tablet as disclosed in the WO2019016668.

The prolonged-release tablets are given to healthy volunteers. After specific time intervals blood sample are collected. The sample are collected in tubes containing anticoagulant. The tubes may contain anticoagulants known to the person skilled in the art i.e. EDTA, heparin, citrate and oxalate. Some commercially available tubes are terumo VENOSAFE™ containing NaF/citrate buffer)/Na2EDTA; NaF/Na-heparin; and NaF/K2oxalate. Sarstedt tubes containing NaF/citrate; NaF/Na2EDTA; and K2EDTA. Specifically, in the present invention, the sample were collected in tubes containing K2EDTA as anticoagulant.

Due to the rapid metabolism of Betahistine into 2-PAA by MAO, Betahistine is instable in blood but can be stable in plasma when monoamino oxidase inhibitor (MAOI) is used along with highly controlled low temperature. The monoamino oxidase inhibitor selected from rasagiline, selegiline and safinamide. Specifically the monoamino oxidase inhibitor is selegiline. More specifically selegiline is added to the tube containing sample and anticoagulant.

As Betahistine has a low molecular weight and high polarity, chromatography separation of Betahistine is also very difficult. To achieve low picogram detection and chromatography separation, in one of the embodiment the present invention provides use of ion pairing reagent. There are various ion pairing reagent are known in the literature i.e. di-n-propylammonium acetate, di-n-butylammonium acetate, di-n-amylammonium acetate, di-n-hexylammonium acetate, heptafluorobutyric acid (HFBA), trifluoroacetic acid (TFA), ammonium trifluoroacetic acid (ATFA), pentafluoropropionic acid, heptafluoro-n-butyric acid, nonafluorovaleric acid, undecafluorohexanoic acid, tridecafluoroheptanoic acid and pentadecafluorooctanoic acid.

Not all the ion pairing reagent known to the person skilled in the art gives similar result. Ion pairing reagent according to the present invention is heptafluorobutyric acid (HFBA). Ion pairing regent is used in mobile phase. Specifically, the ion pairing reagent is used in mobile phase B and in reconstitution solution. Specifically, heptafluorobutyric acid is used as ion pairing reagent in mobile phase B and in reconstitution solution.

The method of present invention uses LC-MS/MS for analysis of Betahistine in human plasma.

The bioanalytical method is performed as follow:

Betahistine stock and its dilution preparation:
- Dissolve around 2 mg Betahistine standard in water to produce the stock solution of concentration between 380000000 to 420000000 pg/mL of Betahistine.
- Dilute the stock solution with water to acquire stock dilution of about 2500000 pg/mL of Betahistine.
- Prepare the spiking solution in water.

Internal standard (ISTD) stock and its dilution preparation:
- Dissolve around 1mg Betahistine D3 standard in water to produce the stock solution of concentration between 475000 to 525000 ng/mL of Betahistine D3.
- Dilute the stock solution with water to acquire about 2500 ng/mL of intermediate ISTD dilution of Betahistine D3.
- Dilute the intermediate ISTD with water to acquire about 5 ng/mL of ISTD dilution of Betahistine D3.

Critical Solution used:
- Sellegiline solution prepared in 0.9% Sodium chloride solution, used to prepare buffered plasma.
- HFBA in 0.1 % formic acid in water: Acetonitrile (80:20) for reconstitution of dry sample.
- HFBA in 0.1 % formic acid in water used as Mobile phase B.

Sample Preparation:
Processing condition: ice-cold water bath maintained at or below 4.0°C till samples loading into cartridges.

- Aliquot 500 µL of each sample into pre-labeled tubes.
- Add 50µL of ISTD dilution to each tube except blank sample and add 50 µL of water in to standard blank/blank qc/ext blank samples and vortex for complete mixing of contents.
- Add 500 µL of water to each tubes and vortex to ensure complete mixing of contents.
- Centrifuge the samples at 3345±100 rcf for 5 minutes at = 10ºC temperature.
- Condition the pre-labeled reverse phase polymeric base cartridges with 1.0 mL methanol followed by 1.0 mL of water on SPE manifold applying low pressure.
- After conditioning, load the centrifuged sample, apply low pressure.
- Till loading of samples into cartridges, processing was done in ice-cold water bath maintained at or below 4.0°C temperature.
- Add 1 mL of water and dry the cartridges under full pressure for 5 minutes.
- Elute the cartridges twice with 500 µL of 0.1 % formic acid in acetonitrile into pre-labeled tubes, apply low pressure.
- Evaporate the samples under nitrogen at 40 ± 2 ºC to dryness.
- Cross check the labels of the tubes against auto sampler vials/96 well collection plate before sample transfer.
- Transfer the contents into appropriate vials/96 well collection plate for analysis on LC-MS\MS

Chromatographic conditions:

Column: C18 100*3 mm
Mobile Phase-A: Acetonitrile
Mobile Phase-B: HFBA in 0.1% formic acid in water (v/v)
Flow rate: 0.8 mL/minute
Injection volume: 30 µL
Column oven temperature: 35 °C
Sample cooler temperature: 4 °C
Run time: 7.00 Minutes

Mass Spectrometry Parameters:
MRM file Parameters:
Compound Name Polarity m/z Dwell (ms)
Betahistine positive 137.100/94.000 400
Betahistine D3 (ISTD) positive 140.100/94.000 400

Tune Files Parameters:
State File Parameter ID State File Parameters for (Betahistine) State file parameters for ISTD
Collision activate dissociation 6.00 6.00
Curtain gas 40.00 40.00
Nebulizer gas 65.00 65.00
Auxiliary gas 50.00 50.00
Ion spray voltage 2000.00 2000.00
Temperature 500.00 500.00
Entrance potential 10.00 10.00
Declustering potential 40.00 40.00
Collision energy 19.00 19.00
Cell exit potential 15.00 15.00

EXAMPLES

The present invention has been described by way of example only, and it is to be recognized that modifications thereto falling within the scope and spirit of appended claims, and which would be obvious to a person skilled in the art based upon the disclosure herein, are also considered to be within the scope of this invention.

Example 1: Prolonged release tablet composition

Ingredients mg/tablet
Betahistine dihydrochloride 48
Cellulose microcrystalline 230
Citric acid anhydrous 50
Polyvinylpyrrolidone K30 12
Hydroxypropylmethylcellulose
K100M premium CR 374
Lambda carrageenan* 40
Colloidal anhydrous silica 15
Talc 8
Sodium stearyl fumarate 8
785
* - Commercially available as viscarin PH 209

The Prolonged release tablet composition Example 1 was prepared by following the process disclosed in example 1 of WO2019016668.

Example 2: Effect of different ion pairing agent

Different ion pairing agent as stated below table were used in mobile phase for analysis of prolonged release tablet composition of example 1. The result of the different ion pairing agent were as follow:

Reagents Sensitivity Chromatography
Formic acid Comparable sensitivity with HFBA Broad peak shape compare to HFBA
Trifluoroacetic acid (TFA) Sensitivity drastically low -
Ammonium trifluoroacetic acid (ATFA) Sensitivity drastically low Poor chromatography
Heptafluorobutyric acid (HFBA) Good Sharp peak

From the above it can be concluded that not all ion pairing agents known in the art have same sensitivity for Betahistine and not all ion pairing agents known in the art will give same chromatography results.

Example 3: Bioavailability test using bioanalytical method of the invention

The bioavailability test of the prolonged release tablet composition of example 1 was carried out using bioanalytical method of present invention.

A. Design of the study
The trial was carried out as an open label, balanced, randomized, two treatment, two sequence, two period, crossover, comparative bioavailability study of Betahistine dihydrochloride sustained release tablets 48 mg given as single dose and Betaserc® 24 mg tablets twice a day in healthy, adult, human subjects under fasting condition.

B. Samples
The samples used in the bioavailability test are the following:
a) Test sample: Betahistine dihydrochloride sustained release tablets 48 mg of example 1, given as single dose; and
b) Reference Test: Betaserc® 24 mg tablets given as twice a day dose.

C. Test Method
The test and reference samples were administered to the subjects in sitting posture with 240 ± 02 mL of drinking water at ambient temperature. The tablet to be swallowed has to be whole without chewing or crushing.

For test sample:
After overnight fasting of at least 10 hours, subjects were served standardized high fat high calorie vegetarian breakfast, which they were required to consume within 30 minutes. The test sample was to be administered once at 30 minutes after serving standardized high fat high calorie vegetarian breakfast.

For reference sample:
After overnight fasting of at least 10 hours, subjects were served standardized high fat high calorie vegetarian breakfast, which they are required to consume within 30 minutes. The first dose of reference sample product was administered at 30 minutes after serving standardized high fat high calorie vegetarian breakfast. Further, again standardized high fat high calorie vegetarian breakfast was served at 11.5 hours after first dose, which they were required to consume within 30 minutes. Second dose was administered at 12.00 hours (interval between two consecutive doses is 12 hours ± 5 minutes).

Sampling
At initial time and at each time points (as decided by protocol) after administration, blood was collected.

Blood samples were collected through an indwelling intravenous cannula (Venflon) placed in a forearm vein of the subjects. Intravenous indwelling cannula was kept in situ as long as possible by injecting 0.5 mL of normal saline solution to prevent cannula from clogging for collection of all the blood samples during housing.

The blood samples were centrifuged at 3000 ± 100 rcf for 5 minutes below 10°C to separate plasma. The separated plasma was transferred to pre-labelled polypropylene tube and stored upright in a box containing dry ice or in a freezer at a temperature -65 ± 10 °C for interim storage as required.

Finally, the samples were transferred by keeping them in dry ice box and stored in bioanalytical freezer at -65 ± 10 °C until completion of analysis.

Sample details
Samples collected: Plasma in Vacutainers
(Anticoagulant: K2EDTA)
Number of periods: 2
Number of subjects: 16
Sampling schedule For Betahistine: Total 62 Time-points

Test Product (32 Time-points):
pre-dose (0.000) and at 0.167, 0.333, 0.667, 1.000, 1.500, 2.000, 2.500, 3.000, 3.500, 4.000, 4.500, 5.000, 5.500, 6.000, 6.500, 7.000, 7.500, 8.000, 8.500, 9.000, 10.000, 11.000, 12.000, 13.000, 14.000, 15.000, 16.000, 18.000, 20.000, 24.000 and 48.000 hours post-dose.

Reference Product (30 Time-points):
pre-dose (0.000) and at 0.083, 0.167, 0.250, 0.333, 0.500, 0.750, 1.000, 1.250, 1.500, 1.750, 2.000, 3.000, 4.000, 12.000, 12.083, 12.167, 12.250, 12.333, 12.500, 12.750, 13.000, 13.250, 13.500, 13.750, 14.000, 15.000, 16.000, 24.000 and 48.000 hours post-dose.

Standard sample details:
Source of matrix for calibration and quality control samples: Human blank plasma was obtained in-house (Clinical Facility, Lambda Therapeutic Research Limited, Ahemedabad, India)

Screening of matrix: The human plasma used for the preparation of calibration curve standards and quality control samples was found free from significant interference at the retention times and transitions of Betahistine and Betahistine D3 (ISTD).

Source of Standards:
1. The working standard for Betahistine dihydrochloride having Batch No. CS/WS/AAA-0201/02 was procured from Clearsynth labs Ltd, Mumbai, India.
2. The working standard for Betahistine D3 Dihydrochloride having Batch No. CRC-0257-061 was procured from Clearsynth labs Ltd., Mumbai, India.

Sample Preparation for analysis:
Processing condition: ice-cold water bath was maintained at or below 4.0 °C till samples loading into cartridges.

- 500 µL of each samples was aliquoted into pre-labeled tubes.
- 50 µL of ISTD dilution was added to each tube except blank sample and add 50 µL of water in to standard blank samples and vortex for complete mixing of contents.
- 500µL of water was added to each tubes and vortex to ensure complete mixing of contents.
- The samples were centrifuged at 3345±100 rcf for 5 minutes at = 10ºC temperature.
- The pre-labeled reverse phase polymeric base cartridges were conditioned with 1.0 mL methanol followed by 1.0 mL of water on SPE manifold applying low pressure.
- After conditioning, the centrifuged sample were loaded, low pressure was applied.
- 1 mL of water was added and the cartridges were dried under full pressure for 5 minutes.
- The cartridges were eluted twice with 500 µL of 0.1 % formic acid in acetonitrile into pre-labeled tubes, applying low pressure.
- The samples were evaporated under nitrogen at 40 ± 2ºC to dryness.
- The contents were transfered into appropriate vials/96 well collection plate for analysis on LC-MS\MS.

Solution used in the analysis:
1. 5 % Selegiline solution prepared in 0.9% sodium chloride solution were used to prepare buffered plasma
2. Acetonitrile:water (50:50) solution were used for rinsing solution
3. 0.1 % Formic acid in acetonitrile solution were used for elution purpose
4. 0.01% HFBA in 0.1 % formic acid in water:acetonitrile (80:20) were used for reconstitution
5. ISTD dilution of 5ng/mL

Analysis of the prepared samples were done using LC-MS\MS in condition specified below:

Chromatographic conditions:

Column: C18 100*3 mm
Mobile Phase-A: Acetonitrile
Mobile Phase-B: HFBA in 0.1% Formic acid in water (v/v)
Initial Pump B%: 98 %
Initial Divert valve: 1 position
Flow rate: 0.8 mL/minute
Injection volume: 30 µL
Column oven temperature: 35°C
Sample cooler temperature: 4°C
Retention Time : Betahistine around 1.6 min
Betahistine D3 around 1.6 min
Run time: 7.00 Minutes

Mass Spectrometry parameter:

MRM file Parameters:
Compound Name Polarity m/z Dwell (ms)
Betahistine positive 137.100/94.000 400
Betahistine D3 (ISTD) positive 140.100/94.000 400

Tune Files Parameters:
State File Parameter ID State file parameters for A1(Betahistine) State file parameters for ISTD
Collision activate dissociation 6.00 6.00
Curtain gas 40.00 40.00
Nebulizer gas 65.00 65.00
Auxiliary gas 50.00 50.00
Ion spray voltage 2000.00 2000.00
Temperature 500.00 500.00
Entrance potential 10.00 10.00
Declustering potential 40.00 40.00
Collision energy 19.00 19.00
Cell exit potential 15.00 15.00

Table of calibration curve and back calculated concentration of calibration curve standards of accepted analytical runs for Betahistine.

Global Statistics
Betahistine in Human Plasma (pg/mL)
Standard Ids STD1 STD2 STD3 STD4 STD5 STD6 STD7 STD8
Mean 7.547 15.054 98.575 298.767 1005.466 1481.315 1800.72 2026.148
SD ± 0.1011 0.3935 1.5744 1.672 13.0639 14.8465 22.6391 30.7975
Precision (%CV) 1.3 2.6 1.6 0.6 1.3 1 1.3 1.5
Nominal Value 7.536 15.072 99.814 299.293 997.644 1493.479 1799.372 1999.302
Accuracy (% Nominal) 100.1 99.9 98.8 99.8 100.8 99.2 100.1 101.3
N 9 9 9 9 9 9 9 9

The analytical method of the invention provides linear range from 7.5 pg/mL to 2000 pg/mL for Betahistine in human plasma.

Table of values obtained for quality control samples of accepted analytical runs for Betahistine.

Global Statistics
Betahistine in Human Plasma (pg/mL)
Ids HQC MQC LMQC LQC
Mean 1612.175 625.135 112.581 23.558
SD ± 32.9472 11.3662 2.8948 1.0644
Precision (%CV) 2 1.8 2.6 4.5
Nominal Value 1586.058 613.276 110.39 22.078
Accuracy (%) 101.6 101.9 102 106.7
N 18 18 18 18
HQC (High Quality control sample), MQC (Medium quality control sample), LMQC (Low medium quality control sample and LQC (Low quality control sample)

Based on the above global statistics, the method of the invention is precise and accurate for Calibration curve and quality control sample as the results are well within acceptance criteria. Coefficient of determination (r2) is also well within acceptance criteria. Hence by extrapolating this results it can be concluded that the results of clinical sample are accurate.

Bioavailability study result obtained using bioanalytical method of the invention is as below:

Betahistine:
The pharmacokinetic parameters of Betahistine for Test Product-T and Reference Product-R are summarized in the following table:

Descriptive Statistics of Formulation Means for Betahistine (N=31)
Parameters (Units) Mean ± SD
(Untransformed data)

Test Product-T Reference Product-R
Tmax (h)* 0.333 (0.333-0.667) 0.333 (0.167 - 0.667)
Cmax (pg/mL) 416.766 ± 339.6116 371.007 ± 246.7705
AUC0-t (pg.h/mL) 156.063 ± 116.2008 130.841 ± 94.1585
AUC0-8 (pg.h/mL) 170.771 ± 119.5686^ 143.813 ± 94.0112$
?Z (1/h) 3.904 ± 2.2710^ 5.131 ± 2.2144$
t1/2 (h) 0.429 ± 0.8617^ 0.211 ± 0.2754$
AUC_%Extrap_obs (%) 3.736 ± 3.1455^ 3.572 ± 5.9714$
*Tmax is represented in median (min-max) value.
^N=29 and $N=28; Note: Terminal rate constant (?Z) cannot be estimated based on obtained concentration data for subject nos. 1004 (Period-I, R), 1015 (Period-II, R), 1024 (Period-II, T), 1026 (Period-I, R and Period-II, T). Hence, AUC0-8 and other elimination phase dependent parameters cannot be calculated.

Thus, the present invention provides a simple, accurate and precise bioanalytical method for assay of Betahistine in human plasma, wherein the method comprises (a) mixing monoamino oxidase inhibitor (MAOI) and human plasma sample comprising Betahistine. (b) use of Heptafluorobutyric acid (HFBA) as ion pairing reagent in mobile phase, and (c) use of single solid phase extraction (SPE) method for extraction of Betahistine, wherein, the method measures Betahistine itself. ,CLAIMS:We claim:

1. A bioanalytical method for assay of Betahistine in human plasma, wherein the method comprises measurement of Betahistine in human plasma without any surrogate pharmacokinetics parameter.

2. A bioanalytical method for assay of Betahistine in human plasma according to claim 1, wherein the method comprises use of ion pairing reagent in mobile phase.

3. A bioanalytical method for assay of Betahistine in human plasma according to claim 1, wherein the method comprises use of ion pairing reagent in reconstitution solution.

4. A bioanalytical method for assay of Betahistine in human plasma according to claim 2-3, wherein the ion pairing reagent is Heptafluorobutyric acid.

5. A bioanalytical method for assay of Betahistine in human plasma according to claim 4, wherein Heptafluorobutyric acid is used in mobile phase B and in reconstitution solution.

6. A bioanalytical method for assay of Betahistine in human plasma according to claim 1, wherein the method comprises single solid phase extraction process for extraction of Betahistine.

7. A bioanalytical method for assay of Betahistine in human plasma according to claim 1, wherein the method comprises step of mixing monoamine oxidase inhibitor and human plasma sample comprising Betahistine.

8. A bioanalytical method for assay of Betahistine in human plasma according to claim 7, wherein the monoamine oxidase inhibitor is selegiline.

9. A bioanalytical method for assay of Betahistine in human plasma, wherein the method comprises steps of: (a) mixing selegiline and human plasma sample comprising Betahistine, (b) use of single solid phase extraction method for extraction of Betahistine, and (c) using Heptafluorobutyric acid as ion pairing reagent in mobile phase and in reconstitution solution in the assay of Betahistine in human plasma using LC-MS/MS, wherein the method measures Betahistine itself in human plasma without any surrogate pharmacokinetics parameter.

10. A bioanalytical method for assay of Betahistine in human plasma according to claim 1-9, wherein the method measures Betahistine and the method does not involve measurement of Betahistine metabolite 2 pyridyl acetic acid (2-PAA).