Ephedrine/Pseudoephedrine Determination
Dean DuanMilliporeSigma
China application lab Shanghai, China
Ephedrine hydrochloride and pseudoephedrine hydrochloride are the active ingredients in the Chinese traditional medicine Xiao’er Kechuanling oral solution, which can be used to reduce cold, allergic rhinitis, rhinitis and sinusitis caused by nasal congestion symptoms and control bronchial asthma.
This application focuses on the determination of ephedrine HCl and pseudoephedrine HCl. Oral solution samples were distilled to extract ephedrine HCl and pseudoephedrine HCl. Samples were then filtered through Millex® PTFE syringe filters, prior to HPLC-UV analysis using a Discovery® HS C18 column.
The limit of detection (LOD) and the limit of quantitation (LOQ) were 0.002 µg/ mL and 0.006 µg/ mL, respectively, for ephedrine HCl, while it was 0.02 µg/mL and 0.07 µg/ mL, respectively, for pseudoephedrine HCl. The method can be used for determination of ephedrine HCl and pseudoephedrine HCl in Xiao’er Kechuanling oral solution.
Figure 1.Chemical structures of compounds used in the study.
Experimental Conditions | |
---|---|
Column Used: | Discovery® HS C18 25 cm x 4.6 mm,5 µm (568523-U) |
Detection: | Waters e2695, 210 nm |
Mobile phase A: | 10 mM KH2PO4, adjust pH value to 2.3 with H3PO4 |
Mobile phase B: | Methanol |
Gradient: | A : B = 89 : 11, v/v |
Flow Rate: | 1.0 mL/min |
Injection Volume: | 20 µL |
Temp: | 30 °C |
Standard: | Dissolve appropriate amount of ephedrine HCl and pseudoephedrine HCl in mobile phase to obtain a solution containing 5 µg/mL of ephedrine HCl and 3 µg/mL of pseudoephedrine HCl |
Sample: | Accurately weigh 5 mL of oral solution into a 500-mL round-bottomed flask. Add into 120 mL of 10% sodium hydroxide, shake well, distill, collect about 95 mL distillate into a 100-mL volumetric flask containing 5 mL of 10 mM KH2PO4 (adjust pH value to 2.3 with H3PO4). Make up the tick mark with water. Filter through an appropriate 0.45 µm filter membrane. |
Pressure drop: | 130 bar (1872 psi) |
Figure 2.Chromatogram of ephedrine HCl and pseudoephedrine HCl standard solution.
Figure 3.Chromatogram of Xiao’er Kechuanling oral solution.
Figure 4.Chromatogram of solvent blank.
No | Compound | Retention Time (min) | Resolution | Theoretical Plates | Tailing Factor |
---|---|---|---|---|---|
1 | ephedrine HCl | 18.7 | -- | 19214 | 1.2 |
2 | pseudoephedrin HCl | 20.5 | 3.4 | 19491 | 1.1 |
Specificity: Inject reference solution and determine the retention time of desired analyte in presence of other component like impurities. | |||||
---|---|---|---|---|---|
No | Compound | Retention Time (min) | Area (%) | Tailing Factor | RRT |
1 | ephedrine HCl | 18.7 | 63.4 | 1.2 | 1.0 |
2 | pseudoephedrine HCl | 20.5 | 36.6 | 1.1 | 1.1 |
Standard repeatability (5 µg/mL), ephedrine HCl | |
---|---|
STD 1 | 213014 |
STD 2 | 214449 |
STD 3 | 213476 |
STD 4 | 216079 |
STD 5 | 211415 |
Mean | 1547859 |
Standard Deviation | 1729.3 |
RSD (%) | 0.1 |
Standard repeatability (3 µg/mL), pseudoephedrine HCl | |
---|---|
STD 1 | 123123 |
STD 2 | 124686 |
STD 3 | 124313 |
STD 4 | 124316 |
STD 5 | 122888 |
Mean | 1327102 |
Standard Deviation | 803.6 |
RSD (%) | 0.1 |
Linearity (area mAU*min) - ephedrine HCl | |
---|---|
Conc. (µg/mL) | Mean Area |
0.10 | 3251 |
0.20 | 7629 |
0.40 | 16270 |
0.5 | 20589 |
1.0 | 36282 |
2.0 | 79273 |
4.0 | 164205 |
5.0 | 213014 |
6.0 | 249607 |
Figure 5.Calibration curve of ephedrine HCl.
LOD and LOQ - ephedrine HCl | |
---|---|
Conc. (µg/mL) | Mean Area (mAU*min) |
0.10 | 3251 |
0.20 | 7629 |
0.40 | 16270 |
0.50 | 20589 |
STEYEX | 24.1 |
Slope | 43315.6 |
LOD (ppm) | 0.002 |
LOQ (ppm) | 0.006 |
Linearity (area mAU*min) - pseudoephedrine HCl | |
---|---|
Conc. (µg/mL) | Mean Area |
0.06 | 2158 |
0.12 | 4591 |
0.24 | 10025 |
0.30 | 12021 |
0.60 | 20081 |
1.2 | 46035 |
2.4 | 97117 |
3.0 | 123123 |
4.0 | 172293 |
Figure 6.Calibration curve of pseudoephedrine HCl.
LOD and LOQ - pseudoephedrine HCl | |
---|---|
Conc. (µg/mL) | Mean Area (mAU*min) |
0.06 | 2158 |
0.12 | 4591 |
0.24 | 10025 |
0.30 | 12021 |
STEYEX | 272.9 |
Slope | 41932.6 |
LOD (ppm) | 0.02 |
LOQ (ppm) | 0.07 |
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