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Validated HPLC method for the determination of enantiomeric impurity of l-Ephedrine Sulfate.

Introduction

Ephedrine (EP) is a sympathomimetic amine commonly used as a stimulant, appetite suppressant, concentration aid, decongestant, and to treat hypotension associated with anaesthesia. Ephedrine is similar in structure to the synthetic derivatives amphetamine and methamphetamine. Chemically, it is an alkaloid derived from various plants in the genus Ephedra (family Ephedraceae). It is most usually marketed in the l-Ephedrine hydrochloride and sulfate forms, Active pharmaceutical ingredient (API)

A stimulant used in the treatment of asthma and respiratory ailments. Ephedra sinica, a herb containing ephedrine, is included in some herbal supplements used by athletes and others to accelerate fat loss and enhance feelings of physical well-being. These supplements are sometimes promoted as 'fat burners'. There is no conclusive scientific evidence that ephedra-containing supplements improves athletic performance

Chemistry

Ephedrine exhibits optical isomerism and has two chiral centres. By convention the enantiomers with opposite stereochemistry around the chiral centres are designated ephedrine, while pseudoephedrine has same stereochemistry around the chiral carbons. That is, (1R,2R)- and (1S,2S)-enantiomers are designated pseudoephedrine; while (1R,2S)- and (1S,2R)-enantiomers are designated ephedrine. The isomer which is marketed is (-)-(1R,2S)-ephedrine. [14] As with other phenylethylamines, it is also somewhat chemically similar to methamphetamine, although the amphetamines are more potent and have additional biological effects [7]

Ephedrine may also be referred to as: ([alpha]R)- [alpha]-[(1S)-1(methylamino)ethyl]benzenemethanol, [alpha]-[l-(methylamino)ethyl]benzyl alcohol, or Lerythro-2-(methylamino)-l-phenylpropan-l-ol. [15] Ephedrine sulfate has a melting point of 248[degrees]C-252[degrees]C, (C10H15NO)2.H2S04 (2:1 salt) CAS No.[134-72-5], Molecular weight 428.54 [8,10,12].

Manufacture

Although Ephedrine occurs naturally as an alkaloid in certain plant species (for example, as a constituent of extracts from the ephedra species, also known as Ma Huang, in which it occurs together with other isomers of ephedrine), the majority of Ephedrine produced for commercial use is derived from yeast fermentation of dextrose in the presence of benzaldehyde. In this process, specialized strains of yeast (typically a variety of Candida utilis or Saccharomyces cerevisiae) are added to large vats containing water, dextrose and the enzyme pyruvate decarboxylase (such as found in beets and other plants, inter alia). After the yeast has begun fermenting the dextrose, the benzaldehyde is added to the vats and in this environment the yeast convert the precursor ingredients to l-Phenylacetylcarbinol (l-PAC). l-PAC is then chemically converted to ephedrine via reductive amination. The bulk of Ephedrine is produced by commercial pharmaceutical manufacturers in India and China, where economic and industrial conditions favor the mass production of Ephedrine for export.

This article describes developments and validations of a new analytical method, which can detect quantify trace levels of d-EPS in l-EPS.

[FIGURE 1 OMITTED]

Experimental

Materials and Methods

l-EPS and d-EPS were synthesized by Synthetic organic Chemistry group. Both th compounds were characterized [5] for their identity and purity, their enantiomer purity was monitored by specific rotation using polarimeter and were purified ti equal opposite values for specific rotation were obtained. HPLC grade n-Hexan Analytical reagent grade Absolute Ethanol and Diethylamine were obtained fro Merck, India.

HPLC system used was Shimadzu 2010 AHT and Agilent 1200 series. Shimadz system comprised of degasser, quaternary pump, auto injector, column oven an UV/Vis detector. The signal was acquired and processed using LC-solution softwa and Agilent-1200 series system comprised of degasser, quaternary pump, au injector, and column compartment and variable wavelength detector. The system w controlled through EZ Chrome Elite version 3.2.1.

Preparation of solutions and chromatographic conditions

Preparation of solutions System suitability solution (SS)

Accurately weigh about 15mg + 1mg each of l-ephedrine (l-EP) and d-ephedrine (( EP) in to a 10 ml volumetric flask, dissolve and dilute to volume with mobile phase.

Reference solution Stock impurity solution

Accurately weigh 25mg + 2mg of d-ephedrine in a 100 ml volumetric flask, dissolve and dilute to volume with mobile phase.

Reference solution R

Accurately weigh about 66mg + 5mg of l- ephedrine sulfate standard in a 20ml volumetric flask, add 2ml of 1N Sodium hydroxide and dissolve, pipette 10ml of the mobile phase and 1ml of the Stock impurity solution and shake well. Separate the organic layer; add about 500mg of anhydrous sodium sulfate shake well, filter and inject.

Test solution

Accurately weigh about 66mg + 5mg of the test (l-EPS) in a 20ml volumetric flask, add 2ml of 1N Sodium hydroxide and dissolve, pipette 10ml of the mobile phase and shake well. Separate the organic layer; add about 500mg of anhydrous sodium sulfate shake well, filter and inject.

Chromatographic conditions:

The chromatographic column used was a Chiralpak AD-H 4.6 mm X 250 mm with 5|l particle size. Mobile phase consist of a mixture 970 ml of n-Hexane, 30 ml of absolute Ethanol and 1 ml of diethylamine and degas by sonication for 5 minutes. Flow rate of mobile phase was 2.0 ml/min. Column was maintained at 25[degrees]C and column eluent was monitored at 254 nm. Injection volume was 20 ul.

System suitability

Performance of the method was determined by injecting resolution mixture (1.5 mg/ml of l-EP and 1.5 mg/ml of d-EP). Method performance criteria were resolution (R) between two entiomer peaks should be not less than 2.0 [9, 11, and 13].

Linearity

Linearity of response for l-EPS was determined in the range of 0.02 to 0.1 mg/ml (0.4 % to 2.0% of the assay solution concentration i.e. 0.05 mg/ml) The % RSD for linearity solution is not more than 10.0% (L1 - L5).There should be no non-linear trend at the ends of the plotted fitted line. Correlation coefficient (R2) is NLT 0.99. [1-3]

Limit of detection (LOD) and limit of quantification (LOQ)

LOD and LOQ of d-EPS was determined by Signal-to-Noise method. Solutions of isomer was prepared in the range of 0.05% with respect to test and 0.16% with respect to test respectively and injected in six times. The % RSD for LOQ solution must not be higher than 10.0%. The signal-to-noise ratio (S/N) in LOQ solution should be about 10:1 for d-EPS. The signal-to-noise ratio (S/N) in LOD solution should be about 3:1 for d-EPS. [1-3]

Precision, accuracy and ruggedness

Precision of the method was determined by injecting six different preparations and determining % RSD of impurity (d-EPS) values. Accuracy of the method was determined by recovery studies. d-EPS was spiked in pre-analyzed sample of l-EPS and its percent recovery was determined. Ruggedness of the method was determined by performing quantification of d-EPS on two different HPLC systems (HPLC system used was Shimadzu 2010 AHT and Agilent 1200 series) and columns by two analysts. [1-3]

Results and Discussion

The objective of this work was to develop a precise and accurate method to determine enantiomeric purity in l-EPS. Various options were attempted to develop such method. Resolution of 3.3 was obtained by using Mobile phase consist of a mixture 970 ml of n-Hexane, 30 ml of absolute Ethanol and 1 ml of diethylamine with Chiralpak AD-H 4.6 mm X 250 mm stationary phase. This method was further validated for estimation of d-EPS (enantiomeric impurity) in l-EPS bulk drug. As mentioned above application of chiral mobile phase additive give acceptable resolution required to quantify presence of d-EPS in l-EPS.

[FIGURE 2 OMITTED]

Fig 2: HPLC Chromatogram of d-ephedrine (d-EP) and l-ephedrine (l-EP). Chromatograms with peak of d-EP, Retention time 8.6 minutes and l-EP, Retention time of 10.50 minutes. A representative chromatogram showing resolution of derivatives of enantiomers is shown in Fig-2. An excellent resolution (R = 3.3) between the two peaks respectively. The described method was found to be linear for d-EPS in the range of 0.02 to 0.1 mg/ml (0.4 to 2.0% of the assay solution concentration i.e. 0.05 mg/ml). The % RSD for linearity solution is between 0.16% to 0.62% respectively. There is no non-linear trend at the ends of the plotted fitted line, Correlation coefficients ([R.sup.2]) was 1.0. LOD and LOQ for d-EPS was determined by Signal-to-Noise method. LOD and LOQ for d-EPS was found to be 0.04 % (S/N=4:1) with respect to l-EPS drug matrix and 0.16 % (S/N=12:1) with respect to l-PES drug matrix respectively. The % RSD for LOQ solution is 1.84%. The method found to be Precise for the impurity d-EPS detected RSD=1.75% , accurate for the amount spiked and amount found of d-EPS are shown in Table-1 and rugged as content of d-EPS was did not deviate significantly on two systems with overall relative standard deviation 0.9%.

References

[1] ICH [Validation of Analytical Procedures: Methodology (Q2R1)], International Conference on Harmonization, Food and Drug Administration, USA, November 1996 and November 2005.

[2] Reviewer Guidance Validation of Chromatographic Methods, Center for Drug Evaluation and Research (CDER), 1994.

[3] USP-27 Validation of compendial methods <1225> page no. 2622 - 2625

[4] ICH [Impurities in New Drug Substances (Q3A(R2)], International Conference on Harmonization, Food and Drug Administration, USA Current Step 4 version dated 25 October 2006.

[5] S. Ahuja, K. Mills Alsante, Handbook of Isolation and Characterization of Impurities in Pharmaceuticals, Elsevier Science, San Diego, 2003.

[6] Hyun MH, Koo HJ, Jin JS, Lee W. Liquid chromatographic resolution of racemic amines and amino alcohols on a chiral stationary phase derived from crown-ether. J Liq Chromatogr Rel Technol 2000;23:2669-82.

[7] US Patent 6,495,529

[8] British pharmacopoeia 1993, BP 1993 (Vol-I) page 247-248, S47 infra red spectrum and BP 1993 (Vol-II) page895-897

[9] Ph. Eur. method 2.2.46 Appendix III Chromatographic separation technique

[10] United states pharmacopoeia, USP-27; NF 22 the national formulary official from January 1, 2004. (Asian edition) official monographs Ephedrine and its salts page.707-710

[11] USP-27 chromatography <621> page no. 2272-2284

[12] Indian pharmacopoeia (government of India ministry of health & family welfare) IP 1996 volume -I page 282-285 Volume -II (P-Z) S-35 infra red spectrum. published by the controller of publications, Delhi, IP-1996 Ephedrine Salt

[13] Indian pharmacopoeia IP-1996 Appendix 4.3 page no.A67-A68

[14] Edited by Reynolds JEF, ed (1989). Martindale: The complete drug reference

(29th edition ed.). London: Pharmaceutical Press. ISBN 0-85369-210 6.Budavari S, editor. The Merck Index: An encyclopedia of chemicals, drugs, and biologicals, 12th edition. Whitehouse Station: Merck.

(1) K. Gokulakrishnan and (2) K Balamurugan

(1) Head, Department of Chemistry

(1) E-mail: nandhu_gokul@yahoo.com

(2) Prist University, East Campus, Thanjavur--Pin-613403. Tamilnadu, India.

(2) E-mail: kbalmurugan@hotmail.com
Table 1: Recovery of d-EPS.

 Amount      Amount     Recovery     Mean     RSD
added (%)   Found (%)      (%)                (%)

              0.43        99.58
  0.39        0.44       101.89     101.12    1.32
              0.44       101.89
              0.84       101.68
  0.79        0.84       101.68     101.68    0.00
              0.84       101.68
              1.04       101.85
              1.04       101.85
  0.98        1.05       102.83     102.18    0.49
              1.05       102.83
              1.04       101.85
              1.04       101.85
              1.63       100.78
  1.58        1.65       102.02     101.81    0.93
              1.66       102.64
              2.04       101.73
  1.97        2.03       101.23     101.56    0.28
              2.04       101.73

RSD: Relative standard deviation
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Article Details
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Title Annotation:high performance liquid chromatography
Author:Gokulakrishnan, K.; Balamurugan, K.
Publication:International Journal of Applied Chemistry
Article Type:Report
Geographic Code:1USA
Date:Jan 1, 2010
Words:1875
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