Isolation, Characterization of Unknown Impurity in Brimonidine Tartrate by Auto Purifier, NMR, LC/MS/MS and HPLC

 

Shivaji Jadhav1,2, Gajanan Mandawad2, Namdeo Dhokale3, Shankaraiah Konda3*

1Indoco Remedies Ltd., TTC Industrial Area, Rabale, Navi Mumbai - 400701, Maharashtra.

2Department of Chemistry, Maharashtra Udaygiri College, Udgir - 413517, Maharashtra.

3Research Center in Chemistry, K. J. Somaiya College, Kopargaon - 423601, Maharashtra.

*Corresponding Author E-mail: kondasg@rediffmail.com

 

ABSTRACT:

The four impurities in Brimonidine Tartrate bulk drug substances were detected by newly developed gradient reverse phase high performance liquid chromatography (HPLC) method. These impurities were named as Brimonidine Impurity- A, B, C, and D. The Brimonidine Impurity-A, Brimonidine Impurity-B, Brimonidine Impurity-C were process impurities, while Brimonidine Impurity-D is degradation impurity. The degradation Brimonidine Impurity-D was isolated by semi preparative HPLC. Structural elucidation of unknown impurities was carried out by means of (conformed by) spectral studies like IR, NMR and Mass synthesized authentic compounds.

 

KEYWORDS: Brimonidine Tartrate, Impurities, Isolation, Characterization.

 

 

1. INTRODUCTION:

Impurities in pharmaceuticals are the unwanted chemicals that remain with the Active Pharmaceutical Ingredients (API). Brimonidine, chemically 5-bromo-N-(4, 5-dihydro-1H imidazol-2-yl) quinoxalin-6-amine is α2-adrenoreceptor agonist and numerous scientific articles have been published on its biological activity as α2-adrenoceptor agonist1-3. Brimonidine is majorly used in the treatment of open-angle glaucoma, ocular hypertension and rosacea4. Brimonidine is a clonidine derivative that is administered ocularly to lower Intraocular Pressure (IOP) in patients with ocular hypertension or open angle glaucoma. Elevated IOP has long been recognized as a major factor for human glaucoma5.

 

 

The numerous analytical methods such as reversed-phase LC to quantify the brimonidine tartrate in eye drops6,7, and synthesis and detection of potential impurity of brimonidine tartrate by mass spectrometry method8-9 have been published. Since the impurity profile study of any pharmaceutical substance is a crucial part of process development, it was felt necessary to develop a reliable mass compatible method for identification and quantitative determination of impurities in brimonidine tartrate. The four impurities in bulk drug substances were detected by in-house HPLC method, out of which three were process related impurities and one belongs to degradation impurity. A comprehensive study was undertaken for the identification of these impurities using LC/MS/MS followed by their synthesis and isolation and further characterization by various spectroscopic techniques. It also deals with the validation of a new stability indicating HPLC method for quantitative determination of these impurities.

 

2. EXPERIMENTAL:

2.1. Materials and reagents:

Samples of Brimonidine tartrate were obtained from Indoco Laboratories Ltd., Chemical Research Division, Navi Mumbai, India. HPLC grade Acetonitrile, Methanol, Potassium Dihydrogen Phosphate, Sodium hydroxide, Water Milli Q and Trifluoro acetic acid was purchased from Merck India Limited.

2.2. 1. Synthesis of reference material:

BRT Impurity-A and BRT Impurity-B are stating material of BRT while BRT-C is process impurity.  BRT-D is degradation impurity and need to be synthesized.

 

2.2.2. Synthesis of BRT-D:

Degradation impurity D is derived from 5.0g Brimonidine tartrate in methanol by treating with 10 mL of 30% H2O2 reflux for 4-5hrs and resulting reaction mixture was subjected to auto purifier consisting of 2525 binary gradient pump, a 2487UV detector and 2767 sample manager (Waters milford USA). An Inertsil ODS (100mm x 30mm x 5μm) (G.L. Science Japan) was used for preparative isolation. The 0.1% Trifluoro acetic acid in water and acetonitrile with time gradient programme T (min)%A: 0/85, 5/85, 15/60, 16/10, 18/10, 20/85, 22/85. The flow rate was maintained at 25mL/min. The reaction mixture was injected. The injection volume was 4.0mL and the UV detection was monitored at 248nm. The impurity was eluted at about 12.6min. The fractions were collected between retention times 12.4min. and 13.0min. The purity of the isolated fractions was checked by analytical HPLC method in section 2.3. The fraction showing the presence of impurity above 99.0% were mixed together and concentrated to dryness under high vacuum. The HPLC purity of the isolated impurity was found to be above 99%. This isolated solid impurity was used for spectral characterization without any further purification.

 

2.3. High Performance Liquid Chromatography (HPLC):

Samples were analyzed on a Waters Alliance 2695 separation module equipped with 2487 UV detector. A C18 column (Inertsil ODS-3 250cm×4.6cm i.e., 5µ particles) was used for chromatographic separations. The mobile phase consisting of A: Dissolved 4.08g of potassium dihydrogen phosphate, in 1000ml water and adjust pH 4.5 with 0.1N Sodium hydroxide or 0.1% ortho phosphoric acid. B: Acetonitrile and Methanol (100:400 V/V), with a timed gradient programme T (min)/%B: 0/15, 5/15, 15/40, 20/60, 30/60, 35/15, 45/15 and a flow rate of 1.0mL per min was used. The injection volume was 10µL and the detector wavelength was fixed at 248nm. The column was maintained at 25°C temperature throughout the analysis.

 

2.4. Liquid chromatography–tandem mass spectrometry (LC/MS/MS):

The MS and MS/MS studies were performed on LCQ Advantage (Thermo Electron, San Jose, CA) ion trap mass spectrometer. The source voltage was maintained at 6.0kV, APCI vaporizer temperature at 270°C and capillary temperature at 2500C. Nitrogen was used as both sheath and auxiliary gas. The mass to charge ratio was scanned across the range of m/z 110–700. MS/MS studies were carried out by keeping normalized collision energy at 40% and an isolation width of 8amu. The HPLC consisted of Finnigan Surveyor quaternary gradient pump with a degasser, an auto sampler, a PDA detector and column oven. A C18 column (Inertsil ODS-3, 250cm × 4.6cm i.d., 5µ particles) was used for separation. The mobile phase consisting of A: 1000ml water pH adjusted to 2.75 with Trifluoro acetic acid: ACN (940:65) and B: Acetonitrile, with timed gradient programme of T (min)/%B: 0/0, 5/0, 15/20, 25/25, 27/0, 30/0, and a flow rate of 1.5mL per min was used.

 

2.4.1. Isolation and structural elucidation of impurity:

Liquid phase degradation of Brimonidine tartrate was carried out and degradation samples were analyzed using HPLC method. From degradation study it was found that the desired impurity was generated about 3- 4% (area normalization) during peroxide degradation. The experimental details are mentioned in Table-1.

 

2.5. NMR spectroscopy:

The 1H and 13C NMR spectrum of the synthesized and isolated impurity was recorded on Varian 400MHz instrument. The 1Hand 13C chemical shift values were reported on the δ scale (ppm) relative to TMS.

 

3. RESULTS AND DISCUSSION:

3.1. Detection of impurity by IR Spectroscopy:

The IR spectrum for isolated impurities was recorded in the solid state as KBr powder dispersion using Perkin-Elmer spectrum on FT-IR spectrometer.

 

3.2. Detection of impurity by HPLC:

HPLC analysis using the method described in Section 2.3 revealed the presence of four peaks at RRT 0.85, 1.72, 2.05 and 2.5 with respect to principle peak. The target peak under study is marked as impurities. The typical chromatogram highlighting the retention time of this impurity.

 

3.2. Identifcation of impurity by LC/MS/MS:

A mass spectrometry compatible HPLC method, as described in Section 2.3 is used to detect impurity. The mass spectrum obtained for Brimonidine tartarate and four impurities showed a protonated molecular ion (M+H)+ at m/z 294, 222.08, 334.17, 213.13 and 310.  The spectral data obtained from MS/MS. The mass spectrum of Impurities showed protonated molecular ion peak at m/z 222.08, 334.17, 213.13, 310 which was identified as mass of BRT A, B, C, and 310.

            

Brimonidine Impurity-A                          Brimonidine Impurity-B

                        

Brimonidine Impurity-C                          Brimonidine Impurity-D

Brimonidine Impurity-A:

 

 

           m/z = 214.109                                      m/z = 214.109                                                                        m/z = 171.067

 

Brimonidine Impurity-B:

 

 

                                                                       m/z = 310.030                                                     m/z = 223.982

Brimonidine Impurity-C:

 

 

 

                                                                                                   m/z = 233.982                                       m/z = 144.056

Brimonidine Impurity D

 

 

                                                                                               m/z = 310.030                                         m/z = 223.982

 

 

                                                                   m/z = 310.030                                 m/z = 310.030                                              m/z = 293.003

 

 

 

Table-1: Structural elucidations of BRT Impurity-D

Sr. No.

Position

Integration

δ PPM

D2O Exchange

Multiplicity

13C δ PPM

1

1

1 H

8.97

8.97

d

146.6

 

 

 

 

 

 

 

2

2

1 H

8.87

8.87

d

140.83

3

3

-

-

-

-

-

4

4

-

-

-

-

140.72

5

5

-

-

-

-

144.462

6

6

-

-

-

-

-

7

7

-

-

-

-

109.755

8

8

-

-

-

-

155.58

9

9

1 H

8.76

8.73

d

128.72

10

10

1 H

8.05

8.08

d

125.085

11

11

1 H

8.581

-

s

-

12

12

1 H

7.74

-

d

-

13

13

-

-

-

-

139.8

14

14,17

2 H

7.94

-

s

-

15

15,

2 H

3.44

3.45

q

39.568

16

16

2 H

3.00

3.00

q

39.568

 

NMR:

The 1H, 13C and D2O measurements of the isolated impurities and BRT were performed on a AVANCE 400 (Bruker, Fallanden, Switzerland). The impurity BRT C and D was isolated by semi-preparative HPLC and taken for NME spectral analysis. In 1H NMR spectra 17 protons appeared in the upfield region (δ 0.76-5.49 ppm) and 11 in the downfield region (delta 0.76-5.49 ppm). 1H and 13C Spectrum taken together showed two set of additional ethyl group as compared to BRT, concurrently absence of Cyclohexyl 1- chloroethyl carbonate group. This confirms that one set of additional ethyl group in BRT Iimpurity-D forming ethyl after replacing methyl group and another set of extra ethyl group is attached to tetrazole moiety as presumed by MS n fragmentation, considering existence of annular tautomeric forms, 2D NOESY, HMBC and HSQC NMR experiments were carried out. 2D NOESY experiment showed in space correlation of methyl group at H34 and H26. Both NOESY and HMBC predicted that the ethyl group attached to the N32 of tetraole group. Complete proton and carbon position assignment compared with BRT impurity D is given in Table 1. To the best of our knowledge, this is a novel impurity.

 

4. CONCLUSION:

In the summary, a new mass compatible HPLC method was developed for separation of one unknown impurity in Brimonidine tartrate bulk drug sample. This impurity was identified by LC/MS analysis. Characterization of the impurity was carried by synthesis, isolation followed by spectroscopic analysis. The newly developed HPLC method has been validated as per regulatory guidelines; it can be conveniently used for the quantitative determination of related substances in Brimonidine tartrate bulk drug sample. The method was found to be specific, accurate and precise, and can be used for the routine analysis as well as to monitor the stability studies.

 

5. ACKNOWLEDGEMENTS:

The authors wish to thank Indoco Remedies Laboratories, Mumbai for providing required facilities. The authors also thankful to Principal, K. J. Somaiya College Kopargaon for providing necessary facilities.

 

6. REFERENCES:

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5.      Morrison JC, Elevated intraocular pressure and optic nerve injury models in the rat. J. Glaucoma., 2005; 14(4):315-17.

6.      Burke J, Schwartz M, Preclinical evaluation of brimonidine Surv. Ophthalmol. 1996; 41(S1), S9-S18

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8.      Mahajan M, Tiwari R, Method Development and Validation of Brimonidine Tartrate by High Performance Liquid Chromatography. Asian J. Research Chem. 2011; 4(10): 1591-1593

9.      Mussie EW, Ruiz G, Wijono M, Wheeler LA, Neuroprotection of retinal ganglion cells by brimonidine in rats with laser-induced chronic ocular hypertension. Investig. Ophthalmol. Vis. Sci., 2001; 42(12):2849-2855

 

 

Received on 05.08.2025      Revised on 27.08.2025

Accepted on 12.09.2025      Published on 30.09.2025

Available online from October 07, 2025

Asian J. Research Chem.2025; 18(5):327-330.

DOI: 10.52711/0974-4150.2025.00050

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