Enantiomeric Analysis of 

Ephedrines and Norephedrines

S.M. Wanga,b

R.J. Lewisb

D. Canfieldb

T.L. Liabc

R.H. Liubc

a Central Police University

Taoyuan, Taiwan

b Civil Aerospace Medical Institute

Federal Aviation Administration

Oklahoma City, OK 73125

c Department of Justice Sciences

University of Alabama at Birmingham

Birmingham, AL 35294

April 2005

Final Report

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iii

ACKNOWLEDGMENTS

The authors are thankful to the financial support provided by the (Taiwanese) National Bureau of Controlled Drugs, Department of Health (DOH92-NNB-1005), and the provision of a research environment 

by the U.S. Federal Aviation Administration’s Civil Aerospace Medical Institute (Oklahoma City, OK), 

which greatly facilitated the preparation of this manuscript.

1

ENANTIOMERIC ANALYSIS OF EPHEDRINES AND NOREPHEDRINES

INTRODUCTION

“Chirality” is currently a topic at the forefront of academic research, as evidented by the award of the 2001 

Noble Prize in Chemistry to “three scientists who devised 

techniques for catalytic asymmetric synthesis — the use 

of chiral catalysts to accelerate the production of singleenantiomer compounds for pharmaceutical use and a wide 

range of other applications. ”(1) In the pharmaceutical 

industry, drug firms are actively involved in developing 

new drugs as single enantiomers and in carrying out 

“racemic switches” — redeveloping racemic mixture 

drugs as single enantiomers — resulting in a significant 

increase in the percentage of drugs marketed as single 

enantiomers.(2)

Enantiomeric analysis of abused drugs is also an 

important issue in forensic laboratories. Data resulting 

from enantiomeric analysis can (a) provide information 

for sentencing guidance for certain drug-related offenses; 

(b) assist in drug-related investigations; and (c) determine 

whether the drug of concern is derived from a controlled 

substance. For example, ephedrine and pseudoephedrine (ψ-ephedrine) are common over-the-counter (OTC) 

pharmaceuticals. They are also frequently used as adulterants in packaging drugs of abuse.(3) (–)-Ephedrine 

has been a popular precursor for illicit manufacturing of 

(+)-methamphetamine.(4,5) Investigation of clandestine 

laboratory activities reported (6,7) the use of ephedra 

plant (Ma Huang) material for methamphetamine 

manufacturing; (–)-ephedrine and (+)-ψ-ephedrine in 

this plant are extracted for conversion to methamphetamine in these illicit manufacturing processes. Thus, 

the identification of ephedrine and ψ-ephedrine, and 

their enantiomeric composition in methamphetamine 

samples, may help identify the drug’s precursor material 

and provide valuable information to the investigation 

process. Also of significant analytical concern is the reported false methamphetamine identification in urine 

specimens due to excessive consumption of ephedrine 

and ψ-ephedrine.(8)

Enantiomeric analysis of abused drugs in the authors’ laboratories date back to 1981, mainly involving 

gas chromatographic and nuclear magnetic resonance 

spectrometric approaches.(9–13) More recent studies 

utilized liquid chromatography and capillary electrophoresis.(14,15) Concerned with the presence of ephedrine-related compounds in OTC cold remedies and its 

implications in sport drug testing (16), this study was 

conducted to develop a method that can be effectively 

used to determine the enantiomeric compositions of the 

following structurally related compounds: ephedrines, 

Ψ-ephedrine, norephedrines (phenylpropanolamine, 

or PPA), norpseudoephedrine (nor-Ψ-ephedrine, or 

cathine), and cathinones. Methods thereby developed 

were then applied to selected OTC cold remedies to 

detect the presence and enantiomeric compositions of 

these compounds.

EXPERIMENTAL 

Standards and reagents

R(+)-Cathinone, S(–)-cathinone, S,R(+)-ephedrine, 

R,S(–)-ephedrine, S,S(+)-Ψ-ephedrine, R,R(–)-Ψ-ephedrine, S,R/R,S(±)-norephedrine, Internal standard (S,R(+)-

ephedrine-d3, 1 mg/mL in methanol) were purchased from 

Cerilliant Int. Co. (Austin, TX); S,S(+)-nor-Ψ-ephedrine 

(1 mg/mL) was purchased from Sigma Co. (St. Louis, 

MO). The structures of these compounds are shown in 

Figure 1.

Chiral derivatization reagents and their sources are as 

follows: (–)-α-methoxy-α-trifluoromethylphenylacetic 

(MTPA), (S)-(–)-N-(trifluoroacetyl)-prolyl chloride (lTPC) (Aldrich: St. Louis, MO); 2,3,4,6-tetra-O-acetylβ-D-glucopyranosyl isothiocyanate, R-(+)-α-phenylethyl 

isocyanate, 2,3,4-triacetyl-α-D-arabinopyranosyl isothiocyanate (Fluka Chemie Gmbh: Industriestrasse, Buchs, 

Switzerland). Achiral derivatization reagents and their 

sources are: 9-fluorenylmethyl chloromate, N-(phenylseleno)-phthalimide, N,O-bis(trimethylsilyl)-acetamide 

(BSA), pentafluoropropionic anhydride (PFPA), helptafluorobutyric anhydride (HFBA) (Aldrich: St. Louis, 

MO); 4-carboethoxyhexafluorobutyryl chloride (4-CB) 

(Lancaster: Windham, NH).

Nineteen readily available OTC cold remedies (13 

syrup, 6 capsule) were purchased from local drug stores 

in the greater Taipei area.

Sample preparation

Typical extraction, derivatization, and GC-MS analysis 

studies utilized 2 mL of standard mixtures or specimens. 

Standard mixtures were prepared to contain 1000 ng of 

each analyte following the general procedure described 

below. Standards obtained from the suppliers (typically 

1 mg/mL in methanol) were first diluted to 10 μg/mL 

(in ethanol). 100 μL of each standard was then taken and 

mixed into 2 mL of drug-free syrup.
2 

The preparation of OTC samples was as follows. Those 

in syrup forms were diluted (typically diluting 100 μL to 

2 mL), while those in capsule forms were emptied and 

dissolved into 10 mL of blank syrup with further dilution 

(typically diluting 20 μL to 2 mL).

To determine extraction efficiency, the following procedure was used to derive the amount of the analytes 

resulting from the process without the extraction step. A 

mixture containing all analytes of interest was prepared by 

mixing 100 μL each of the diluted standards (10 μg/mL) 

in a clean tube. The mixture was dried under nitrogen and 

then processed in parallel with other standard mixtures 

that had completed the extraction step.

Derivatization procedure

Standard mixtures and OTC specimens in aqueous 

solutions were extracted and derivatized following either a 

one-step or two-step procedure as described below. Using 

l-TPC as example, the one-step procedure involved mixing 

2 mL sample, 100 μL internal standard ((+)-ephedrined3

, 10 μg/mL), 0.5-mL saturated K2CO3 solution, 50 μL 

l-TPC, and 6 mL ethyl acetate for 10 min. The mixture 

was then centrifuged (5 min), followed by removing 

the upper layer to a clean tube that was dried under a 

nitrogen stream. The residue was typically reconstituted 

with 200-μL ethyl acetate of which 1 μL was used for 

each GC-MS analysis.

Using MTPA derivatization as an example, the two-step 

process was carried out as follows. Typically, the internal 

standard, 2 mL standard mixture (or specimen), and 0.5 

mL saturated K2CO3 solution were mixed for 30 sec. The 

mixture was then extracted with 6-mL ethyl acetate by 

shaking (10 min), followed by centrifugation (5 min). 

The upper layer was transferred into a clean tube and 

dried under nitrogen. For the derivatization step, the 

residue was added 50 μL N,N-dicyclohexycarbodiimide 

and 100 μL MTPA. The reaction mixture was thoroughly 

mixed, then incubated at 70oC for 20 min. This same 

two-step procedure was used when HFBA was used for 

derivatization, except that 1 mL of 2 N NaOH, instead 

of 0.5 mL saturated K2CO3 solution, was used prior to 

the addition of ethyl acetate for extraction.

GC-MS analysis

GC-MS analysis was performed on a HP 5890 Series 

II GC interfaced to an HP 5971 MS (Agilent: Palo Alto, 

CA). Two columns used in this study were: 25 m HP 

5MS (0.20 mm ID, 0.33 μm film thickness) and 60 m 

HP 5MS (0.25 mm ID, 0.25 μm film thickness) from 

Agilent (Wilmington, DE). Helium carrier gas flow rate 

was 1.0 mL/min. The injector and GC-MS interface 

temperatures were maintained at 250 and 280oC, respectively. Temperature of the GC oven was programmed 

using different parameters for the analysis of products 

derived from different derivatization reagents (Table 1). 

For the 60 m column, a typical GC-MS run took 30 

min or less.

The MSD was initially operated under full-scan mode 

to derive the retention time and full-scan mass spectrum 

information for each analyte. This information was then 

used to identify each analyte in standard mixtures and 

OTC specimens. Full-scan mass spectra were further used 

for the selection of ions suitable for use in selected ion 

monitoring (SIM) mode.

RESULTS AND DISCUSSION

Resolution

As shown in Section 2.1, a total of 11 derivatization 

reagents (5 chiral and 6 achiral) were included in this 

study. MTPA was found to be the most effective chiral 

derivatization reagent, allowing complete base-line resolution of the 10 structurally closely related compounds of 

interest shown in Figure 2. (–)-Cathine was not available 

for this study; however, it should have been resolved, were 

it included in the mixture.

Shown in Figure 3 are the mass spectra of (+)-Ψ-norephedrine (A), (+)-norephedrine (B), (+)-Ψ-ephedrine 

(C), (+)-Ψ-ephedrine (D), and (+)-ephedrine-d3 (E) (all 

as MTPA derivatives). Mass spectra of the corresponding 

(–)-somers are practically indistinguishable and, therefore, 

are not shown.

Derivatization products resulting from the commonly used l-TPC also result in good resolution of the 

analytes, with the exception of (–)-ephedrine and (–)-

Ψ-ephedrine. Furthermore, ions that may be used for 

designating the analytes and their deuterated analogs are 

less characteristic.

When the determination of analytes’ enantiomeric 

compositions is not needed, HFBA-derivatization was 

found very effective (Fig. 4). Analysis time can be further 

reduced by increasing the column temperature following 

the elution of cathine and norephedrine (peaks 1 and 2 in 

Fig. 4). Mass spectra of representative compounds with 

HFBA derivatization are shown in Figure 5.

Evaluation of analytical parameters

Common analytical parameters, such as limits of 

detection and quantitation (LOD and LOQ) and extraction efficiency, have been studied. Evaluations were 

performed on MTPA and HFBA derivatizations using 

ephedrine as the exemplar compound. Results listed in 

Table 2 were established using the criteria and procedure 

described below.

Commonly adapted criteria were used to confirm the 

presence of a specific analyte in a test sample, i.e., ions 
3

monitored for a specific analyte have to present at an acceptable retention time (±2%) with acceptable intensity 

ratios (±20%) of that established by a standard. The LOD 

was defined as the lowest concentration of a standard solution meeting the above criteria, while LOQ was defined as 

the lowest concentration of a standard solution that met 

these criteria and with an observed analyte concentration 

that is within ±20% of the targeted value.

A series of standard solutions with the following concentrations of ephedrine were used for LOD and LOQ 

evaluations: 2000, 1000, 500, 250, 100, 80, 60, 40 ng/mL. 

Applying the criteria described above, the method’s LOD 

and LOQ were determined to be 60 and 80 ng/mL for 

both HFBA and MTPA derivatives. 

Application to the analysis of common OTC cold 

remedies

As reported in an earlier study (16), various ephedrinerelated compounds were found in readily available OTC 

cold remedies. Attempts to correlate the occurrences and 

concentrations of these compounds in OTC remedies 

with the analytical findings derived from testing athletes 

during sport-competition events have not been conclusive. 

With this in mind, the authors thought an additional 

dimension of information (enantiomeric composition) 

may help studies of this nature. Thus, various chiral and 

achiral derivatization approaches were explored, of which 

the most effective ones were applied to the analysis of a 

limited number of OTC cold remedies (from 19 manufacturers). Preliminary data shown in Table 3 are promising, and further studies will be pursued and applied to a 

comprehensive list of OTC remedies, selected prescription 

medicines, and relevant urine specimen sets.

CONCLUSION

An effective methodology has been established for 

the analysis of the following structurally related compounds and their enantiomers: cathinone, ephedrine, 

Ψ-ephedrine, norephedrine, and nor-Ψ-ephedrine. 

Using MTPA as the derivatization reagent, the resulting 

products can be baseline resolved by a 60 m HP 5MS 

capillary column. HFBA is effective when enantiomeric 

compositions are not needed. Preliminary application 

studies have also shown great potentials in providing 

an additional dimension of information (enantiomeric 

compositions) for source-tracing studies.

REFERENCES

(1) Long J, Yarnell A, Eds. Chemistry Nobel honors 

Knowle, Noyori, Sharpless for chiral synthesis. 

C&EN, Oct. 15, 2001, 5.

(2) Stinson SC. Chiral pharmaceutical, C&EN, Oct. 15, 

2001, 79–97.

(3) Noggle FT, Clark CR. Liquid chromatographic analysis of samples containing cocaine local anesthetics 

and other amines. J Assoc Off Anal Chem. 1983; 

66: 151–7.

(4) Ely RA, McGrath DC. Lithium-ammonia reduction of 

ephedrine to methamphetamine: An unusual clandestine synthesis. J Forensic Sci. 1990; 35: 720–3.

(5) Noggle FT, DeRuiter J, Clark CN. Liquid chromatographic determination of enantiomeric composition 

of methamphetamine prepared from ephedrine and 

pseudoephedrine. Anal Chem. 1988; 58: 1643–8.

(6) Andrews KM. Ephedra’s role as a precursor in the 

clandestine manufacture of methamphetamine. J 

Forensic Sci. 1995; 40: 551–560.

(7) Hutchinson K, Andrews KM. The use and availability of ephedra products in the United States. 

Microgram. 1995; 28: 256–63.

(8) Department of Health and Human Services, Notice 

to All DHHS/NIDA Certified Laboratories, Feb. 

4, 1991.

(9) Liu J[R]H, Ku WW. Determination of enantiomeric 

N-trifluoroacetyl-l-prolyl chloride amphetamine 

derivatives by capillary gas-liquid chromatography/

mass spectrometry with chiral and achiral stationary 

phases. Anal Chem. 1981; 53: 2180–7.

(10) Liu J[R]H, Ku WW, Tsay JT, Fitzgerald MP, Kim 

S. Approaches to drug sample differentiation. 

III: A comparative study of the use of chiral and 

achiral capillary column gas chromatography/mass 

spectrometry for the determination of methamphetamine enantiomers and possible impurities. J 

Forensic Sci. 1982; 27: 39–48.

(11) Liu J[R]H, Ku WW, Tsay JT, Fitzgerald MP. 

Separation and characterization of drugs and 

their enantiomers by capillary column gas-liquid 

chromatography/mass spectrometry. J Assoc Off Anal 

Chem. 1983; 66: 1443–6.
4 

(12) Liu J[R]H, Tsay JT. The use of chiral lanthanide 

shift reagents for the nuclear magnetic resonance 

spectrometric determination of amphetamine enantiomers. Analyst. 1982; 107: 544–9.

(13) Liu J[R]H, Ramesh S, Tsay JT, Ku WW, Fitzgerald 

MP, Angelos S, Lins CLK. Approaches to drug 

sample differentiation. II: Nuclear magnetic resonance spectrometric determination of amphetamine 

enantiomers. J Forensic Sci. 1981; 26: 656–63.

(14) Sellers JK, Duffitt GL, Gaiones ML, Liu RH. High 

performance liquid chromatographic analysis of enantiomeric composition of abused drugs. Forensic 

Sci Rev. 1996; 8: 91–109.

(15) Iwanicki RM, Maier K, Zlotnick JA, Liu RH, Kuo 

TL, Tagliaro F, Separation of enantiomeric ephedrine and pseudoephedrine — High pressure liquid 

chromatography and capillary electrophoresis. J 

Forensic Sci. 1999; 44: 470–4.

(16) Tseng YL, Hsu HR, Kuo FH, Shieh MH, Chang 

CF. Ephedrine in over-the-counter cold medicines 

and urine specimens collected during sport competitions. J Anal Toxicol. 2003; 27: 359–65.
5

FIGURES AND TABLES

H OH

H NHMe

CH3

HO H

NHMe H

CH3

HO H

H NHMe

CH3

H OH

NHMe H

CH3

 () (+) (+) () 

Ephedrine Pseudoephedrine 

H OH

H NH2

CH3

HO H

H2N H

CH3

HO

H NH2

CH3

H H OH

H2N H

CH3

 () (+) (+) () 

 Norephedrine (PPA) Pseudonorephedrine (Cathine) 

O

NH2

H O

NH2

H

(+) () 

Cathinone 

Figure 1. Structures of ephedrine and structurally related compounds. 

Figure 1. Structures of ephedrine and structurally related compounds.
6 

20.00 20.50 21.00 21.50 22.00 22.50 23.00 23.50 24.00 24.50 25.00

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

Time-->

Abundance

TIC: 1501001.D

 20.18

 20.62

 21.34 21.85

 22.07

 23.08

 23.47

 23.74 24.00

Figure 2. Ion chromatogram of MTPA-derivatives: (+)-Cathinone (1), ()-cathinone (2), (+)-norephedrine 

(3), ()-norephedrine (4), (+)-nor-�-ephedrine (5), (+)-ephedrine-d3 (6), (+)-ephedrine (7), ()-ephedrine 

(8), ()-�-ephedrine (9), and (+)-�-ephedrine (10) (all as MTPA-derivatives). 

7

2

 3 

4

 5 

6

1

8

9 10
7

Figure 3. Mass spectra of (+)-�-norephedrine (A), (+)-norephedrine (B), (+)-�-ephedrine (C), (+)-

ephedrine (D), and (+)-ephedrine-d3 (E) (all as MTPA-derivatives). 

Figure 3. Mass spectra of (+)-Ψ-norephedrine (A), (+)-norephedrine (B), (+)-Ψ-ephedrine (C), (+)-

ephedrine (D), and (+)-ephedrine-d3 (E) (all as MTPA-derivatives).
8 

22.00 22.50 23.00 23.50 24.00 24.50 25.00 25.50 26.00 26.50 27.00

0

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

550000

600000

650000

700000

Time-->

Abundance

TIC: 1901003.D

 22.13

 22.25

 23.85

 25.04

Figure 4. Ion chromatogram of HFBA-derivatives: Cathine (1), (±)-norephedrine (2), (±)-ephedrine (3), 

(±)-�-ephedrine (4), (+)-ephedrine-d3 (5) (all as HFBA-derivatives). 

5 3

2 4

1
9

Figure 5. Mass spectra of (+)-�-norephedrine (A), (+)-norephedrine (B), (+)-�-ephedrine (C), (+)-

ephedrine (D), and (+)-ephedrine-d3 (E) (all as HFBA-derivatives). Figure 5. Mass spectra of (+)-Ψ-norephedrine (A), (+)-norephedrine (B), (+)-Ψephedrine (C), (+)-ephedrine (D), and (+)-ephedrine-d3 (E) (all as HFBA-derivatives).
10

TABLES 

Table 1. Gas chromatograph oven temperature programming parameters for the analysis of analytes 

resulting from three derivatization reagents. 

Derivatization Starting Hold Rate End Hold Rate End Hold 

reagent (

o

C) (Min) (oC/Min) (oC) (Min) (oC/Min) (oC) (Min) 

HFBA 60 0 5 200 0 25 250  

l-TPC 160 5 5 250     

MTPA 160 0 5 220 1 25 250  

Table 2. Evaluation of common analytical parameters resulting from FHBA and MTPA derivatizations. 

Parameter HFBA MTPA 

Recovery (%)a

 72 ± 4 b 90 ± 7 b

LOD (ng/mL) 60 60 

LOQ (ng/mL) 80 80 

a

 Evaluated using triplicates of 2-mL standard solutions containing 500 ng/mL ephedrine. 

b

 Mean ± standard deviation. 

Table 3. Enantiomeric composition (μg/mL) of the targeted 10 analytes found in 

various cold remedies. 

 Cathinone Norephedrine Nor-�-ephedrine Ephedrine �-Ephedrine 

Sample Derivative (+) () (+) () (+) ()b

 (+) () (+) () 

 2 MTPA  a    0.039 b  c  1.84 0.565  

 HFBA   0.123 1.50 0.850 

 9 MTPA       c  2.39 1.02  

 HFBA    2.33 0.951 

10 MTPA       c  1.36 0.737  

 HFBA    1.27 0.600 

15 MTPA 0.186      c

     

 HFBA      

16 MTPA       c 0.549 0.414 48.1  

 HFBA    4.62 21.9 

17 MTPA       c  0.111 0.089  

 HFBA    0.115 0.043 b

19 MTPA 1.35 26.8 1.19 2130 340  c     

 HFBA  2860 0.940   

 a Below detection limit (60 ng/mL as established for ephedrine). 

b

 Below LOD and LOQ as established for ephedrine. However, distinct chromatographic peaks and mass 

spectra were observed and the listed concentrations were estimated. 

c

 No standard was available; thus, these analytical findings are tentative.