Ephedrine/Pseudoephedrine Determination

Dean Duan^Merck

China application lab Shanghai, China

Section Overview

• Determination of Ephedrine HCL and Pseudoephedrine HCL in Xiao’Er Kechuanling Oral Solution
• Specificity and Repeatability – Ephedrine and Pseudoephedrine
• Calibration Data – Pseudoephedrine HCL
• Conclusion
• Related Materials

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.

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

Table 1. HPLC conditions.

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.

Table 2. Specificity data of ephedrine HCl and pseudoephedrine HCl.

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 and repeatability – ephedrine and pseudoephedrine

Table 3. Specificity data of ephedrine and pseudoephedrine.

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

Table 4. Standard repeatability data of ephedrine.

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

Table 5. Standard repeatability (3 µg/mL), pseudoephedrine HCl.

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

Table 6. Linear range of ephedrine HCl.

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.

Table 7. LOD and LOQ 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

Calibration data – pseudoephedrine HCl

Table 8. Linear range of pseudoephedrine HCl.

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.

Table 9. LOD and LOQ 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

Conclusion

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