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HomeSmall Molecule HPLCEphedrine/Pseudoephedrine Determination

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.

2-D chemical structures (bond line structure) of ephedrine HCl and pseudoephedrine HCl compounds used to determine analytes in Xiao’er Kechuanling oral solution.

Figure 1.Chemical structures of compounds used in the study.

Determination of ephedrine HCl and pseudoephedrine HCl in Xiao’er Kechuanling oral solution

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)
Table 1.HPLC conditions.
Separation of ephedrine HCl and pseudoephedrine HCl standards used to determine analytes in Xiao’er Kechuanling oral solution by HPLC-UV method.

Figure 2.Chromatogram of ephedrine HCl and pseudoephedrine HCl standard solution.

Chromatogram showing peaks of Xiao’er Kechuanling oral solution used to determine analytes in Xiao’er Kechuanling oral solution by HPLC-UV method.

Figure 3.Chromatogram of Xiao’er Kechuanling oral solution.

Chromatographic run of solvent blank in HPLC-UV separation of ephedrine HCl and pseudoephedrine HCl standards used to determine analytes in Xiao’er Kechuanling oral solution.

Figure 4.Chromatogram of solvent blank.

NoCompoundRetention Time (min)ResolutionTheoretical PlatesTailing Factor
1ephedrine HCl18.7--192141.2
2 pseudoephedrin HCl20.53.4194911.1
Table 2.Specificity data of ephedrine HCl and pseudoephedrine HCl.

Specificity and repeatability – ephedrine and pseudoephedrine

Specificity: Inject reference solution and determine the retention time of desired analyte in presence of other component like impurities.
NoCompoundRetention Time (min)Area (%)Tailing FactorRRT
1ephedrine HCl18.763.41.21.0
2pseudoephedrine HCl20.536.61.11.1
Table 3.Specificity data of ephedrine and pseudoephedrine.
Standard repeatability (5 µg/mL), ephedrine HCl
STD 1213014
STD 2214449
STD 3213476
STD 4216079
STD 5211415
Mean1547859
Standard Deviation1729.3
RSD (%)0.1
Table 4.Standard repeatability data of ephedrine.
Standard repeatability (3 µg/mL), pseudoephedrine HCl
STD 1123123
STD 2124686
STD 3124313
STD 4124316
STD 5122888
Mean1327102
Standard Deviation803.6
RSD (%)0.1
Table 5.Standard repeatability (3 µg/mL), pseudoephedrine HCl.
Linearity (area mAU*min) - ephedrine HCl
Conc. (µg/mL)Mean Area
0.103251
0.207629
0.4016270
0.520589
1.036282
2.079273
4.0164205
5.0213014
6.0249607
Table 6.Linear range of ephedrine HCl.
Calibration curve of ephedrine HCl to measure the analyte in Xiao’er Kechuanling oral solution using Discovery® HS C18 column.

Figure 5.Calibration curve of ephedrine HCl.

LOD and LOQ - ephedrine HCl
Conc. (µg/mL)Mean Area (mAU*min)
0.103251
0.207629
0.4016270
0.5020589
STEYEX24.1
Slope43315.6
LOD (ppm)0.002
LOQ (ppm)0.006
Table 7.LOD and LOQ of ephedrine HCl.

Calibration data – pseudoephedrine HCl

Linearity (area mAU*min) - pseudoephedrine HCl
Conc. (µg/mL)Mean Area
0.062158
0.124591
0.2410025
0.3012021
0.6020081
1.246035
2.497117
3.0123123
4.0172293
Table 8.Linear range of pseudoephedrine HCl.
Calibration curve of pseudoephedrine HCl to measure the analyte in Xiao’er Kechuanling oral solution using Discovery® HS C18 column.

Figure 6.Calibration curve of pseudoephedrine HCl.

LOD and LOQ - pseudoephedrine HCl
Conc. (µg/mL)Mean Area (mAU*min)
0.062158
0.124591
0.2410025
0.3012021
STEYEX272.9
Slope41932.6
LOD (ppm)0.02
LOQ (ppm)0.07
Table 9.LOD and LOQ of pseudoephedrine HCl.

Conclusion

The method can be used for ephedrine HCl and pseudoephedrine HCl in Xiao’er Kechuanling oral solution.

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