Validated HPLC method for the determination of enantiomeric impurity of d-Pseudoephedrine sulfate.Introduction
Pseudoephedrine (commonly abbreviated as PES) is a sympathomimetic amine commonly used as a decongestant. The salts pseudoephedrine hydrochloride and pseudoephedrine sulfate [8,10,12] are found in many over-the-counter preparations either as single-ingredient preparations, or more commonly in combination with antihistamines, paracetamol and/or ibuprofen. Consumers often refer to it by a product which contains pseudoephedrine, such as Sudafed, the trademark for a common brand of pseudoephedrine hydrochloride in North America.
Unlike antihistamines, which modify the systemic histamine-mediated allergic response, pseudoephedrine only relieves nasal congestion commonly associated with colds or allergies. The advantage of oral pseudoephedrine over topical nasal preparations, such as oxymetazoline, is that it does not cause rebound congestion (rhinitis medicamentosa); however, it is more likely to cause adverse effects including hypertension
Pseudoephedrine is a phenethylamine, and an isomer of ephedrine. Pseudoephedrine is a chiral molecule, meaning it occurs as in both "left-handed" and "right-handed" configurations which are not super imposable.
Pseudoephedrine is the International Nonproprietary Name (INN) of the (1S,2S)-diastereomer of ephedrine (which has 1R,2S- configuration). Other names are (+)-Pseudoephedrine and d-Pseudoephedrine.
l-Pseudoephedrine, also known as (-)-(1R, 2R)-pseudoephedrine or (-)-Pseudoephedrine, is the optical isomer of d-Pseudoephedrine. It has fewer side-effects, fewer central nervous system (CNS) stimulatory effects, does not reduce to d-methamphetamine (which is the enatiomer used as a recreational drug) and yet it retains its efficacy as a decongestant. However, the patent holder for l-Pseudoephedrine (Pfizer/Warner-Lambert) has not yet sought or received government approval for its sale to the public 
Although Pseudoephedrine 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 pseudoephedrine 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 Pseudoephedrine via reductive amination.
The bulk of Pseudoephedrine is produced by commercial pharmaceutical manufacturers in India and China, where economic and industrial conditions favor the mass production of pseudoephedrine for export.
This article describes developments and validations of a new analytical method, which can detect quantify trace levels of l-PES in d-PES.
[FIGURE 1 OMITTED]
Materials and Methods
d-PES and l-PES were synthesized by Medicinal Chemistry group. Both the compounds were characterized  for their identity and purity, their enantiomeric purity was monitored by specific rotation using polarimeter and were purified till equal opposite values for specific rotation were obtained. HPLC grade n-Hexane and Isopropyl alcohol, Analytical reagent grade Absolute Ethanol and Diethylamine. All HPLC and Analytical reagent grade were obtained from Merck, India.
HPLC system used was Shimadzu 2010 AHT and Agilent 1200 series. Shimadzu system comprised of degasser, quaternary pump, auto injector, column oven and UV/Vis detector. The signal was acquired and processed using LC-solution software and Agilent-1200 series system comprised of degasser, quaternary pump, auto injector, and column compartment and variable wavelength detector. The system was 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 [+ or -] 1mg each of d-Pseudoephedrine and l-Pseudoephedrine into a 10 ml volumetric flask, dissolve and dilute to volume with mobile phase.
Stock impurity solution
Accurately weigh 25mg [+ or -] 2mg of l-Pseudoephedrine in to a 100 ml volumetric flask, dissolve and dilute to volume with mobile phase.
Reference solution R
Accurately weigh about 66 mg [+ or -] 5 mg of d- PES 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.
Accurately weigh about 66 mg [+ or -] 5mg of the test (d-PES) 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. solution of about 6.6 mg/ml was used for quantification purpose.
The chromatographic column used was a Chiralpak AD-H 4.6 mm X 250 mm with 5[micro] particle size. Mobile phase consist of a mixture 980 ml of n-Hexane, 10 ml of absolute Ethanol, 10 ml of Isopropyl alcohol 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[degree]C and column eluent was monitored at 254 nm. Injection volume was 20 [micro]l.
Performance of the method was determined by injecting resolution mixture (1.5 mg/ml of d-PES and 1.5 mg/ml of l-PSE). Method performance criteria were resolution (R) between two entiomer peaks should be not less than 2.0, tailing factor(TF) not more than 3.0 [9,11,13].
Linearity of response for l-PES 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 ([R.sup.2]) is NLT 0.99. [1-3]
Limit of detection (LOD) and limit of quantification (LOQ)
LOD and LOQ of l-PES was determined by Signal-to-Noise method. Solutions of isomer was prepared in the range of 0.04% 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 l-PES. The signal-to-noise ratio (S/N) in LOD solution should be about 3:1 for l-PES. [1-3]
Precision and accuracy and ruggedness
Precision of the method was determined by injecting six different preparations and determining % RSD of impurity (l-PES) values. Accuracy of the method was determined by recovery studies. l-PES was spiked in pre-analyzed sample of d-PES and its percent recovery was determined. Ruggedness of the method was determined by performing quantification of l-PES 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 of d-PES. Various options were attempted to develop such method. Resolution of 3.4 was obtained by using Mobile phase consist of a mixture 980 ml of n-Hexane, 10 ml of absolute Ethanol, 10 ml of Isopropyl alcohol and 1 ml of diethylamine with Chiralpak AD-H 4.6 mm X 250 mm stationary phase. Since chiral center is present in the side chain of d-PES. This method was further validated for estimation of l-PES (enantiomeric impurity) in d-PES bulk drug. As mentioned above application of chiral mobile phase additive give acceptable resolution required to quantify presence of l-PES in d-PES.
A representative chromatogram showing resolution of derivatives of enantiomers is shown in Fig-2. An excellent resolution (R = 3.4) between the two peaks along with tailing factor (TF) for d-PES and l-PES was 2.7 and 2.5 respectively.
[FIGURE 2 OMITTED]
The described method was found to be linear for l-PES 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 1.25% to 4.5% respectively. There is no non-linear trend at the ends of the plotted fitted line, Correlation coefficients ([R.sup.2]) was 0.996.
LOD and LOQ for l-PES was determined by Signal-to-Noise method. LOD and LOQ for l-PES was found to be 0.04 % (S/N=4:1) with respect to d-PES drug matrix and 0.16 % (S/N=11:1) with respect to d-PES drug matrix respectively. The % RSD for LOQ solution is 5.7%.
The method found to be Precise for the impurity l-PES detected %RSD=1.25%, accurate for the amount spiked and amount found of l-PES are shown in Table-1 and rugged as content of l-PES was did not deviate significantly on two systems with overall relative standard deviation 0.7%.
 ICH [Validation of Analytical Procedures: Methodology (Q2R1)], International Conference on Harmonization, Food and Drug Administration, USA, November 1996 and November 2005.
 Reviewer Guidance Validation of Chromatographic Methods, Center for Drug Evaluation and Research (CDER), 1994.
 USP-27 Validation of compendial methods <1225> page no. 2622 - 2625
 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.
 S. Ahuja, K. Mills Alsante, Handbook of Isolation and Characterization of Impurities in Pharmaceuticals, Elsevier Science, San Diego, 2003.
 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.
 US Patent 6,495,529
 European Pharmacopoeia, 5th ed., 2004 and British pharmacopoeia 1993, volume-1(International edition) Pseudoephedrine HCl page no. 563/ Volume-II page no.108. Infrared reference spectra S115
 Ph. Eur. method 2.2.46 Appendix III Chromatographic separation technique
 United states pharmacopoeia, USP-27; NF 22 the national formulary official from January 1, 2004. (Asian edition) official monographs Pseudoephedrine and its salts page no. 1598-1602
 USP-27 chromatography <621> page no. 2272 - 2284
 Indian pharmacopoeia (government of India ministry of health & family welfare) IP 1996 volume -II (P-Z) published by the controller of publications, Delhi, IP-1996 Pseudoephedrine HCl page no. 641-642, infrared reference spectra S-87
 Indian pharmacopoeia IP-1996 Appendix 4.3 page no.A67 - A68
(1) K. Gokulakrishnanm and (2) K. Balamurugan
(1) Head, Department of Chemistry
(2) Prist University, East Campus, Thanjavur--Pin- 613403. Tamilnadu, India.
(1) E-mail: email@example.com
(2) E-mail: firstname.lastname@example.org
Table 1: Recovery of L-PES. Amount Amount Recovery RSD added (%) Found (%) (%) Mean SD (%) 0.39 0.36 92.44 92.44 0.0 0.0 0.36 92.44 0.36 92.44 0.79 0.89 113.06 113.91 0.73 0.64 0.90 114.33 0.90 114.33 0.97 1.04 107.14 107.48 1.06 0.99 1.05 108.17 1.06 109.20 1.04 107.14 1.03 106.11 1.04 107.14 1.56 1.71 109.77 111.27 2.06 1.85 1.77 113.62 1.72 110.42 1.99 2.24 112.39 112.72 1.53 6.94 2.28 114.40 2.22 111.39 SD: Standard deviation, RSD: Relative standard deviation