Determination of Ephedra Alkaloids in Urine and Plasma by HPLC-UV: Collaborative Study

Abstract

Ten collaborating laboratories determined the ephedra alkaloid content (ephedrine, pseudoephedrine, norephedrine, norpseudoephedrine, methylephedrine, and methylpseudoephedrine) in 8 blind duplicates of human plasma and urine using high-performance liquid chromatography (HPLC) with UV detection. In addition to negative urine and plasma controls, urine samples were spiked with individual ephedra alkaloids ranging in concentration from about 1 to 5 μg/mL. Plasma samples were spiked with individual ephedra alkaloids ranging in concentration from about 100 to 400 ng/mL. Sample solutions were treated to solid-phase extraction using a strong-cation exchange column to help remove interferences. The HPLC analyses were performed on a polar-embedded phenyl column using UV detection at 210 nm. The ephedra alkaloids were not consistently detected in any of the spiked plasma samples. When ephedra alkaloids were detected in the plasma samples, reproducibility between blind replicate samples was very poor. Repeatability, reproducibility, and accuracy were also very poor for the spiked urine samples. On the basis of these re sults, the HPLC-UV method for the determination of ephedra alkaloids in human urine and plasma is not recommended for adoption as Official First Action.

Ephedra is a shrub-like evergreen plant found in arid regions of Europe, central Asia, and other parts of the world. Major species of ephedra include Ephedra sinica Stapf., E. equisetina Bunge, E. intermedia, and E. distachya. The traditional Chinese medicine Ma Huang is derived from aerial parts of ephedra (1-3), and has been used for the treatment of asthma, bronchial spasms, and as a stimulant and diaphoretic (4). Ephedra is known to contain 6 bioactive alkaloids: ephedrine (EP), pseudoephedrine (PS), methylephedrine (ME), methylpseudoephedrine (MP), norephedrine (NE), and norpseudoephedrine (NP). These alkaloids constitute about 1 to 2.5 wt % of the plant on a dry weight basis, with ephedrine accounting for between 30and 90% of this total (5). The species E. nevadensis, E. antisyphilitica, and E. trifurca, all found in North America, have thus far been found to be free of these alkaloids (6).

EP and PS are available as single-entity or combination prescription and over-the-counter drugs in the United States. NP is a Schedule IV controlled substance requiring a Drug Enforcement Agency (DEA) permit in the United States. In recent years, the number of dietary supplements containing ephedra, either as powdered botanical, or more commonly, as a standardized extract, has increased dramatically. Most of these products are sold as diet aids or energy boosters due to their “thermogenic” effect—the ability to raise the rate at which calories are burned. Often these dietary supplements will also contain caffeine, either synthetic or from botanical extracts, in addition to other ingredients.

There have been a number of severe adverse events associated with the use of ephedra products reported in recent years (7, 8). Often, these adverse events can be attributed to overdoses of ephedra alkaloids, and recently there have been several well-publicized deaths of professional athletes in which ephedra products may have been implicated. Products containing ephedra are currently banned in the National Football League (NFL), the National Hockey League (NHL), and Major League Baseball (MLB).

Because of the health and legal implications associated with the use of products containing ephedra, it is desirable to have a method that can accurately and reproducibly quantify the 6 ephedra alkaloids in human biological (plasma and urine) samples. All 6 ephedra alkaloids are structurally very similar. They are very hydrophilic amine compounds that have poor retention on traditional reversed-phase high-performance liquid chro matography (HPLC) systems. Their basic nature often leads to excessively broad peaks and peak tailing on chromatographic systems. Lastly, they have poor UV absorption above about 210 nm. Currently there are no published HPLC-UV methods for determination of all 6 ephedra alkaloids in biological samples. A method was developed and validated for quantitation of ephedra alkaloids in urine and plasma. This method uses strong-cation exchange (SCX) solid-phase extraction (SPE) as a cleanup step and low-wavelength UV detection.

Collaborative Study

Study Design

An HPLC-UV method, validated in-house by Covance Laboratories (Madison, WI), was sub mitted to 11 laboratories participating in the collaborative study. One laboratory was not able to participate as they did not have the capability to handle biological samples. Each laboratory was sent 8 materials (4 plasma samples and 4 urine samples) as blind duplicates, for a total of 16 samples. One blind duplicate of each matrix was a negative control, containing no ephedra alkaloids at a concentration of >0.500 μg/mL for urine and 50.0 ng/mL for plasma. Each of the remaining samples contained known concentrations of the ephedra alkaloids at 3 different levels. Random identification numbers were assigned to each sample. Each sample was blinded in terms of concentration of ephedra alkaloids.

Collaborators

Eleven laboratories originally agreed to participate in the collaborative study and received materials to conduct the study. Ten laboratories completed the study in the allotted time. Of the 10 laboratories that completed the study, 5 were from the United States, 2 were from Canada, 2 were from Asia, and one was from Europe.

Test Sample Preparation

Source of materials

Biochemed Pharmaceuticals, Inc. (Winchester, VA) supplied the unfortified urine and plasma samples. Both samples came from normal pooled, mixed gender donors. Standards were obtained from commercial sources as described in the methods section.

Preparation and Shipment

The individually prepared test samples, standards, chromatographic column, and SPE cartridges were provided to each collaborative laboratory. The standards and samples were shipped on dry ice to each laboratory with a return receipt document. Collaborators were instructed to store the standards and samples refrigerated (2°–8°C) until use. Each collaborative laboratory prepared their own calibration and sample solutions according to the study protocol provided.

Validated Method Performance (9)

Concentration range

The calibration curves had a range from about 0.25 to 10 μg/mL for each individual ephedra alkaloid. These values correspond to ephedra alkaloid concentrations from about 1 to 40 μg/mL in the samples. The concentration range in the collaborative study was subsequently changed to 0.500 to 10 μg/mL for urine and 50.0 to 1000 ng/mL for plasma.

Validation Data

Recoveries ranged from 69 to 162% for all spiking levels for all ephedra alkaloids. EP recoveries ranged from 93 to 123%, and PS recoveries ranged from 92 to 120%. Table 1 shows the average recoveries for each alkaloid as determined in the single laboratory validation. Table 2 shows the overall precision of the method for the samples fortified at 3 different levels, performed in triplicate.

Table 1.

Single laboratory validation data recovery^a

	Overall recovery, %
Alkaloids	Low spike	Medium spike	High spike
Urine
NE	96.1	95.5	97.4
NP	92.9	93.9	94.0
EP	101	99.7	98.3
PS	109	104	99.7
ME	116	113	94.6
MP	120	113	105
Plasma
NE	89.2	98.2	105
NP	81.9	87.7	101
EP	98.7	105	109
PS	102	102	103
ME	119	114	119
MP	113	110	116

^aNE = Norephedrine; NP = norpseudoephedrine; EP = ephedrine; PS = pseudoephedrine; ME = methylephedrine; MP = methylpseudoephedrine.

Table 2.

Single laboratory validation data precision^a

	Overall precision, % RSD
Alkaloids	Low spike	Medium spike	High spike
Urine
NE	15.4	10.1	4.8
NP	22.7	11.5	6.2
EP	9.1	5.8	3.6
PS	14.1	7.8	3.2
ME	29.3	11.4	17.3
MP	31.2	14.2	4.9
Plasma
NE	4.3	8.0	20.3
NP	8.1	7.1	17.6
EP	8.2	6.0	11.7
PS	7.4	3.7	9.1

^aNE = Norephedrine; NP = norpseudoephedrine; EP = ephedrine; PS = pseudoephedrine; ME = methylephedrine; MP = methylpseudoephedrine.

METHOD

Determination of Ephedra Alkaloids in Human Plasma and Urine by HPLC-UV

The method is applicable to the analysis of ephedra alkaloids (ephedrine, pseudoephedrine, norephedrine, norpseudoephedrine, methylephedrine, and methylpseudoephedrine) in biological fluids such as human plasma and urine.

Principle

The ephedra alkaloids are extracted from human plasma and urine using methanol and then cleaned up using an SCX SPE column. The amounts of ephedra alkaloids present are quantified relative to the recovery of the internal standard (1-methyl-3-phenylpropylamine; MPA) and the individual standards using HPLC with UV detection at 210 nm.

Apparatus

• (a) HPLC system equipped with UV-Vis detector.

• (b) Column.—Phenomenex (Torrance, CA) Synergi Polar RP HPLC, 4.6 × 150 mm, 4 μm particle size.

• (c) Analytical balance.—Readability 0.000 01 g.

• (d) Volumetric flasks.—1000, 100, 50, and 25 mL.

• (e) Volumetric pipets.—0.5, 1, 2, 5, and 10 mL.

• (f) Graduated cylinders.—1000 and 100 mL.

• (g) Mobile phase containers.—1 L.

• (h) Filtration apparatus.—0.45 μm nylon filter.

• (i) Conical polypropylene centrifuge tubes.—15 mL, graduated to read 0.5 mL capped.

• (j) Vortex mixer.

• (k) Ultrasonic bath.

• (l) Syringe filter.—0.45 μm PTFE.

• (m) SCX SPE cartridge containing 500 mg resin.—Phenomenex Strata SCX.

• (n) Autopipettor.—Dispensing 200–1000 μL.

• (o) Benchtop centrifuge.

Reagents

• (a) Deionized water.

• (b) Methanol.—HPLC grade.

• (c) Potassium phosphate, monobasic.—ACS reagent grade.

• (d) Ammonium hydroxide.—ACS reagent grade.

• (e) 85% H₃PO₄.—ACS reagent grade.

Reference Standards

• (a) EP·HCl.—99% (ChromaDex, Santa Ana, CA).

• (b) PS·HCl.—99% (ChromaDex).

• (c) NE.—99% (ChromaDex).

• (d) ME.—98% (ChromaDex).

• (e) MP.—99% (ChromaDex).

• (f) NP.—(1) Cathine HCl, 98% (RBI, a subsidiary of Sigma-Aldrich Chemical Co., Milwaukee, WI). Note: NP is a DEA Schedule IV restricted compound. (2) 1.00 mg/mL (ChromaDex).

• (g) MPA.—97% (Aldrich Chemical Co.).

Preparation of Reagents

• (a) Mobile phase buffer solution.—Dissolve 13.6 g potassium phosphate monobasic (KH₂PO₄) in 1000 mL water. Prepare fresh weekly.

• (b) Mobile phase.—Mix 30 mL methanol with 970 mL mobile phase buffer solution. Filter through a 0.45 μm nylon filter and degas under vacuum. Prepare fresh weekly.

• (c) Diluent.—Mix 30 mL methanol with 970 mL water. Add 1.3 g KH₂PO₄ and stir until all salt is dissolved; then degas. Prepare fresh weekly.

• (d) 50mM H₃PO₄.—Add about 345 μL 85% H₃PO₄ to 100 mL water and mix well. Prepare fresh every 2 weeks.

• (e) 500mM H₃PO₄.—Add about 3.5 mL 85% H₃PO₄ to 100 mL water and mix well. Prepare fresh every 2 weeks.

• (f) SPE diluent solution.—Dilute 5 mL NH₄OH (30%) to 100 mL with water.

• (g) SPE wash solution.—Add 75 mL SPE diluent solution to 25 mL methanol and mix well.

• (h) SPE elution solvent.—Mix 5 mL NH₄OH with 95 mL methanol. Prepare fresh every 2 weeks.

Preparation of Standard, U.S. Laboratories

(a) Stock related alkaloid solution.—Accurately weigh 60 ± 0.2 mg each of EP and PS, and 50 ± 0.2 mg each of NE, ME, and MP into separate 100 mL volumetric flasks. Record exact weights. Add 40 mL methanol and 40 mL diluent, and sonicate until all standards are dissolved (ca 5 to 10 min). Dilute to volume with diluent and mix well. Store solutions in a refrigerator at 2°–8°C and protect from light. Prepare fresh at least monthly.

Accurately weigh 5 mg NP into a 10 mL volumetric flask. Add 4 mL methanol and 4 mL diluent, and sonicate until all standard is dissolved (ca 5 to 10 min). Dilute to volume with diluent and mix well. Store solution in a refrigerator at 2°–8°C and protect from light. Prepare fresh at least monthly.

(b) Stock internal standard solution.—Weigh 50.0 mg MPA into a 100 mL volumetric flask. Dilute to volume with methanol and mix well. Store solution in a refrigerator at 2°–8°C and protect from light. Prepare fresh at least monthly.

(c) Extracting internal standard spiking solution.—Pipet 10.0 mL internal standard stock solution into a 100 mL volumetric flask and dilute to volume with methanol. Store solution in a refrigerator at 2°–8°C and protect from light. Prepare fresh at least monthly.

(d) Calibration standard solutions.—Pipet the indicated volume of stock standard solution in Table 3 into separate 100 mL volumetric flasks. Pipet 2 mL stock internal standard into each flask, and dilute to volume with diluent.

Table 3.

Preparation of calibration standard soultions (United States)

Volume stock standard solution, mL	Final volume, mL	Concn of each ephedra alkaloid, μg/mL	Concn of MPA, μg/mL
0.050 [Level 1]	100	0.25	10
0.100 [Level 2]	100	0.50	10
0.200 [Level 3]	100	1.0	10
0.500 [Level 4]	100	2.5	10
1.00 [Level 5]	100	5.0	10
2.00 [Level 6]	100	10.0	10
Standard blanka	100	0.0	10

^a2 mL methanol + 98 mL diluent.

Note: The linearity standards are stable for at least 2 weeks when stored in ambient laboratory conditions.

Preparation of Standards, International Laboratories

(a) Stock related alkaloid solutions.—Accurately weigh 60 ± 0.2 mg each of EP and PS, and 50 ± 0.2 mg each of NE, ME, and MP into separate 100 mL volumetric flasks. Record exact weights. Add 40 mL methanol and 40 mL diluent, and sonicate until all standards are dissolved (ca 5 to 10 min). Dilute to volume with diluent and mix well. Store solutions in a refrigerator at 2°–8°C and protect from light. Prepare fresh at least monthly.

Quantitatively transfer contents of 1 ampoule NP reference standard solution (1 mg/mL) into a 5 mL volumetric flask. Rinse the ampoule twice with methanol, and transfer the rinsings to the same 5 mL volumetric flask. Dilute to volume with diluent and mix well. Store solutions in a refrigerator at 2°–8°C and protect from light. Prepare fresh at least monthly.

(b) Stock internal standard solution.—Weigh 50.0 mg MPA into a 100 mL volumetric flask. Dilute to volume with methanol and mix well. Store solution in a refrigerator at 2°–8°C and protect from light. Prepare fresh at least monthly.

(c) Extracting internal standard spiking solution.—Pipet 10.0 mL internal standard stock solution into a 100 mL volumetric flask and dilute to volume with methanol. Store solution in a refrigerator at 2°–8°C and protect from light. Prepare fresh at least monthly.

(d) Calibration standard solutions.—Pipet the indicated volume of stock standard solution in Table 4 into separate 100 mL volumetric flasks. Pipet 2 mL stock internal standard into each flask, and dilute to volume with diluent.

Table 4.

Preparation of calibration standard solutions (international)

Volume stock standard solution, mL	Final volume, mL	Concn of NE, EP, PS, ME, and MP, μg/mL	Concn of NP, μg/mL	Concn of MPA, μg/mL
0.050 [Level 1]	100	0.25	0.10	10
0.100 [Level 2]	100	0.50	0.20	10
0.200 [Level 3]	100	1.0	0.40	10
0.500 Level 4]	100	2.5	1.0	10
1.00 [Level 5]	100	5.0	2.0	10
2.00 [Level 6]	100	10.0	4.0	10
Standard blanka	100	0.0	0.0	10

^a2 mL methanol + 98 mL diluent.

Note: The linearity standards are stable for at least 2 weeks when stored in ambient laboratory conditions.

Preparation of Samples

Safety

All biological samples and materials/equipment contacted by the biological samples should be handled with universal procedures and precautions for handling blood samples and human pathogens.

(a) Urine.—Transfer 2 mL urine into a 15 mL polypropylene centrifuge tube. Accurately pipet 2.00 mL extracting internal standard spiking solution into the tube. Note: If spiking is used, add methanol to adjust volume so that the total volume of urine and aqueous fluid is equal to the amount of methanol. Cap the centrifuge tube, and mix on a Vortex mixer for at least 20 s; then centrifuge at 2500 rpm for 10 min or until the supernatant is clear.

(b) Plasma.—Transfer 2 mL plasma into a 15 mL polypropylene centrifuge tube. Pipet 2.00 mL extracting internal standard spiking solution into the tube. Note: If spiking is used, add methanol to adjust volume so that the total volume of plasma and aqueous fluid is equal to the volume of methanol. Cap the centrifuge tube, and mix on a Vortex mixer for at least 20 s. Then centrifuge at 2500 rpm for 10 min or until the supernatant is clear.

(c) SPE.—The procedure uses an SPE column containing 500 mg SCX resin. Use a vacuum manifold designed for SPE. The typical vacuum required to pull the solutions at a reasonable flow through the columns is approximately 5 in. Hg, although this value will depend on the particular columns used, and the amount of particulates in the solution. (1) SPE conditioning step.—Condition an SPE cartridge by first passing 2 mL methanol through the cartridge, followed by 1 mL 50mM H₃PO₄. Do not allow to dry. (2) Loading.—Pipet 2 mL supernatant test solution onto SPE column. Pull solution through column at a rate not exceeding 2 mL per minute. Allow column to be pulled to dryness. Discard the eluate. Run a 2.0 mL blank (methanol) with each set of assays. (3) Column wash.—Wash SPE column by first passing 1 mL 50mM H₃PO₄ solution through column. Allow column to be pulled to dryness. Discard the eluate. Next, pass 2 mL methanol through the column. Allow column to be pulled to dryness. Discard the eluate. Finally, pass 1 mL SPE wash solution through column. Allow column to be pulled to dryness. Discard the eluate. (4) Ephedra alkaloid elution.—Place a 15 mL centrifuge tube below the SPE cartridge. Pipet 1 mL SPE elution solution onto the cartridge and pull the solvent into the tube. Add an other 1 mL SPE elution solution onto the cartridge and pull the solvent into the tube. Add a third 1 mL aliquot of SPE elution so lution onto the cartridge and pull the solvent into the tube. Fill the tube to the 5 mL volume line with 500mM H₃PO₄ and mix well. Transfer a portion to an HPLC autosampler vial.

Note: The standard and test solutions that have been treated by the SPE procedure are stable for at least 1 month when stored at ambient room temperature.

Determination

The standard and test solutions are analyzed using the chromatographic conditions shown in Table 5.

Table 5.

Chromatographic conditions

Column	Phenomenex Synergi PolarRP, 4.6 × 150 mm, 4 μm particle size
Mobile phase	Methanol–100mM KH2PO4 3 + 97 (v/v)
Flow rate	1.5 mL/min
Injection volume	20 μL
Detection	ca 0.10 AUFS at 210 nm
Injection needle wash solvent	Water
Column temperature	25°C
Run time	20 min
Retention times
Marker compound	Retention time, min	Relative retention
Norephedrine	5.7	0.58
Norpseudoephedrine	6.8	0.69
Ephedrine	9.8	1.0
Pseudoephedrine	11.6	1.2
Methylephedrine	15.6	1.6
Methylpseudoephedrine	16.9	1.7
1-Methyl-3-phenylpropylamine	32.0	3.3

System suitability

Repeatability

The relative standard deviation (RSD) of the ephedrine peak area for at least 5 consecutive injections of the 100% linearity standard solution [Level 4 Standard] must not be more than 2.0%. The RSD of each of the related compound peak areas for at least 5 consecutive injections of the 100% linearity standard solution must not be more than 3.0%.

Resolution

The resolution between PS and EP in the working standard solution chromatograms should not be less than 2.0. The resolution between MP and ME in the working standard solution chromatograms should not be less than 1.3.

Tailing

The tailing factor must be no more than 1.5 for all the alkaloids in the linearity standard solution chromatograms.

Linearity

The determination coefficient R² for the regression line (peak area vs concentration) for each ephedra alkaloids must not be less than 0.995. The recovery at each linearity point for each ephedra alkaloid must not be less than 95%. Adjust each standard concentration for its reported purity.

Calculations

Calibration

Plot the average peak area vs concentration (in μg/mL) for each ephedra alkaloid in the linearity standard solution chromatograms, and calculate their linear regressions and determination coefficients. Do not drive the regression through zero.

The amount of analyte found in the sample (urine or plasma) is calculated as follows:

$$\mathrm{A}=\frac{\mathrm{C}\times {\mathrm{V}}_{\mathrm{IS}}\times {\mathrm{C}}_{\mathrm{EIS}}}{{\mathrm{V}}_{\mathrm{b}}\times {\mathrm{C}}_{\mathrm{IS}}}$$

where A = ephedrine-type alkaloid found in test sample (μg/mL); C = concentration (μg/mL) of ephedrine-type alkaloid found in the test samples calculated using the standard curve; V_b = test portion volume (mL, plasma or urine); V_IS = extracted internal standard spiking solution volume (mL); C_EIS = concentration (μg/mL) of extracted internal standard spiking solution; C_IS = concentration (μg/mL) of internal standard in HPLC calibration standards.

Results and Discussion

Results, in micrograms ephedra alkaloid per milliliter sample, for each of the 8 blind replicate samples are presented in Table 6 (for urine) and Table 7 (for plasma). Test samples were given random codes prior to shipment to the collaborators, and then decoded when the results were returned. Table 8 presents a statistical summary of the urine results. Only NP, PS, ME, and MP results for the high spike urine samples, and NP for the middle spike urine sample had a statistically significant number of reported results (≥8 laboratories), and only these values are reported in the table. To use data from at least 8 laboratories, no outliers were removed. For the plasma samples, 6 laboratories did not report detectable levels of any ephedra alkaloids for the 3 fortified levels; therefore, statistical analysis was not performed on these results. Statistical analysis to determine repeatability and reproducibility for the urine samples was performed using the AOAC Interlaboratory Statistical Program 2001 for Blind Replicates (10). Repeatability standard deviations (S_r), reproducibility standard deviations (S_R), repeatability relative standard deviations (RSD_r), reproducibility relative standard deviations (RSD_R), and number of statistical outliers are presented. HORRAT values are also presented in these tables and are calculated as RSD_R (observed)/RSD_R (predicted), where the RSD_R (predicted) is calculated using the equation as follows:

$${\mathrm{RSD}}_{\mathrm{R}}=2{\mathrm{C}}^{-0.1505}$$

where C is the measured analyte concentration in decimal mass units (11). Cochran, Grubbs', and double Grubbs' tests were used to remove statistical outliers where appropriate. All determination coefficients (R²) are greater than 0.998.

Table 6.

Results for blind replicates urine

Results, μg/mL
	Low urine spike		Medium urine spike		High urine spike		Blank
Lab ID	A1	A2	B1	B2	C1	C2	D1	D2
Norephedrine
A	2.54	2.82	3.09	2.86	6.10	6.15	2.19	2.11
B	0.365	0.459	0.630	0.538	3.20	2.14	0.603	ND
C	a	a	a	a	6.62	6.66	a	a
D	2.65	a	3.27	2.64	8.52	9.00	1.94	1.10
E	1.24	1.18	1.50	1.48	3.84	3.90	NDb	ND
F	a	a	a	a	a	a	a	a
G	c	c	c	c	c	c	ND	ND
H	2.30	1.12	1.03	1.00	6.42	4.46	0.200	1.36
I	a	a	a	a	a	a	a	a
J	2.86	2.75	3.84	3.94	6.93	7.57	1.80	1.94
Norpseudoephedrine
A	3.96	3.82	3.63	3.64	3.20	3.22	4.01	3.96
B	1.56	1.24	2.38	1.60	8.65	4.57	6.26	1.39
C	4.72	4.54	4.84	4.94	8.34	8.32	3.73	3.68
D	4.84	a	4.12	4.38	7.48	8.18	3.86	2.34
E	18.8	18.4	19.2	19.4	35.3	35.2	ND	ND
F	a	a	a	a	a	a	a	a
G	0.289	0.354	0.530	0.472	2.58	2.06	ND	ND
H	24.6	20.1	19.8	21.2	4.81	22.0	0.438	24.2
I	a	a	a	a	a	a	a	a
J	3.78	4.16	5.34	5.40	7.50	8.12	2.89	2.56
Ephedrine
A	1.84	1.80	2.35	2.18	6.10	6.18	0.495	0.399
B	1.44	1.53	1.78	1.72	5.92	5.40	1.53	ND
C	a	a	a	a	a	a	a	a
D	a	a	a	a	8.72	8.05	a	a
E	5.06	6.36	6.82	5.41	8.78	8.64	3.56	3.32
F	a	a	a	a	a	a	a	a
G	c	a	a	a	1.86	2.44	ND	ND
H	1.08	2.40	2.88	3.02	4.59	5.97	10.6	0.618
I	a	a	a	a	a	a	a	a
J	4.66	4.44	5.47	5.28	9.35	9.42	3.86	3.00
Pseudoephedrine
A	0.680	0.721	1.12	0.907	4.93	4.04	a	a
B	2.52	3.02	5.55	4.68	8.51	6.37	ND	ND
C	a	a	1.10	1.02	3.83	3.92	a	a
D	a	a	1.46	0.765	5.20	5.16	a	a
E	0.635	0.852	1.01	0.770	4.05	4.00	ND	ND
F	a	a	a	a	a	a	a	a
G	0.208	a	a	a	2.09	1.93	ND	ND
H	0.970	1.20	1.24	1.64	5.38	5.44	ND	1.09
I	a	a	a	a	a	a	a	a
J	1.19	1.07	1.40	1.52	5.12	4.84	0.668	0.168
Methylephedrine
A	3.64	3.63	3.91	3.90	6.84	6.68	3.07	3.02
B	2.58	2.34	3.40	2.80	9.54	5.80	5.40	ND
C	a	a	a	a	3.88	4.32	a	a
D	4.20	a	4.48	2.04	7.57	8.25	0.925	0.820
E	ND	ND	ND	ND	6.24	6.02	ND	ND
F	a	a	a	a	a	a	a	a
G	0.988	0.837	1.16	1.12	2.30	2.48	ND	ND
H	2.86	a	a	a	6.20	4.68	1.62	2.04
I	a	a	a	a	a	a	a	a
J	1.99	2.24	2.32	2.55	6.14	5.99	1.47	1.14
Methylpseudoephedrine
A	11.9	11.6	12.4	11.8	15.4	14.8	10.6	10.8
B	0.762	0.605	0.984	1.08	5.76	5.18	ND	ND
C	a	a	1.52	1.28	5.09	5.16	a	a
D	1.54	a	1.94	1.25	6.32	6.02	a	a
E	ND	ND	ND	ND	4.92	5.29	ND	ND
F	a	a	a	a	a	a	a	a
G	0.546	a	0.856	0.726	1.98	2.04	ND	ND
H	a	a	1.36	0.858	5.42	4.68	a	a
I	a	a	a	a	a	a	a	a
J	2.27	2.15	2.55	2.68	7.12	6.74	1.47	1.24

^aValue below limit of quantitation.

^bND = None detected.

^cInterfering peak prevented quantitation.

Table 7.

Results for blind duplicates plasma

Results, μg/mL
	Low plasma spike		Middle plasma spike		High plasma spike		Blank plasma
Lab ID	E1	E2	F1	F2	G1	G2	H1	H2
Norephedrine
A	NDa	ND	ND	ND	ND	ND	ND	ND
B	ND	ND	ND	ND	ND	ND	ND	ND
C	b	b	b	b	b	b	b	b
D	b	b	b	b	b	b	b	b
E	b	b	b	0.122	0.466	0.462	ND	ND
F	b	b	b	b	b	b	b	b
G	c	c	c	c	c	c	ND	ND
H	b	b	0.362	0.376	0.487	0.582	b	b
I	b	b	b	b	b	b	b	b
J	0.168	0.160	0.218	1.48	0.555	0.440	0.175	0.058
Norpseudoephedrine
A	ND	ND	ND	ND	ND	ND	ND	ND
B	26.1	25.8	28.6	29.8	29.0	32.0	32.0	31.2
C	b	b	b	b	b	b	b	b
D	b	b	b	b	b	b	b	b
E	b	b	0.166	0.287	1.54	1.42	ND	ND
F	b	b	b	b	b	b	b	b
G	b	b	b	b	b	b	ND	ND
H	13.3	14.0	19.4	12.0	17.2	16.4	19.1	12.3
I	b	b	b	b	b	b	b	b
J	0.092	0.078	0.095	0.100	0.232	0.210	0.062	0.068
Ephedrine
A	ND	ND	ND	ND	ND	ND	ND	ND
B	ND	ND	ND	ND	0.856	0.902	ND	ND
C	b	b	b	b	b	b	b	b
D	b	b	b	b	b	b	b	b
E	0.148	0.162	0.166	0.228	0.604	0.616	ND	ND
F	b	b	b	b	b	b	b	b
G	ND	c	ND	c	b	c	ND	ND
H	0.355	b	b	b	0.550	0.514	b	b
I	89	79	4950	4720	51300	49400	ND	ND
J	0.302	0.282	0.292	2.55	0.570	c	ND	0.140
Pseudoephedrine
A	ND	ND	ND	ND	ND	ND	ND	ND
B	6.56	11.2	12.0	8.85	2.78	7.06	1.94	2.75
C	b	b	b	b	b	b	b	b
D	b	b	b	b	b	b	b	b
E	b	b	b	b	0.167	0.188	ND	ND
F	b	b	b	b	b	b	b	b
G	ND	c	ND	b	b	c	ND	ND
H	1.00	1.08	b	b	b	4.24	b	b
I	b	b	b	b	b	b	b	b
J	0.192	0.198	0.255	0.262	0.468	0.402	ND	0.145
Methylephedrine
A	ND	ND	ND	ND	ND	ND	ND	ND
B	ND	ND	ND	ND	1.34	1.36	ND	ND
C	b	b	b	b	b	b	b	b
D	b	b	b	b	b	b	b	b
E	ND	ND	ND	ND	0.264	0.328	ND	ND
F	b	b	b	b	b	b	b	b
G	ND	ND	ND	c	b	c	ND	ND
H	0.691	b	b	b	b	1.88	b	b
I	b	b	b	b	b	b	b	b
J	0.178	0.172	0.178	0.340	0.485	0.430	2.46	0.15
Methylpseudoephedrine
A	ND	ND	ND	ND	ND	ND	ND	ND
B	ND	ND	ND	ND	ND	ND	ND	ND
C	b	b	b	b	b	b	b	b
D	b	b	b	b	b	b	b	b
E	ND	ND	ND	ND	ND	ND	ND	ND
F	b	b	b	b	b	b	b	b
G	ND	ND	ND	c	b	c	ND	ND
H	0.758	0.674	b	b	b	1.44	b	b
I	b	b	b	b	b	b	b	b
J	1.86	2.02	2.49	2.19	2.76	2.04	2.16	2.16

^aND = None detected.

^bValue below limit of quantitation.

^cInterfering peak prevented quantitation.

Table 8.

Statistical analysis of spike urine blind replicates (repeatability and reproducibility)

Sample	Average, μg/g	Sr	RSDr, %	SR	RSDR, %	HORRAT	Recovery, %	No. of labs used
Norpseudoephedrine
Low spike	—	—	—	—	—	—	—	—
Middle spike	7.55	0.41	5.43	7.79	103	8.74	600	8
High spike	10.6	4.42	41.8	11.0	104	9.24	229	8
Pseudoephedrine
Low spike	—	—	—	—	—	—	—	—
Middle spike	—	—	—	—	—	—	—	—
High spike	4.68	0.586	12.5	1.60	34.2	2.70	99.1	8
Methylephedrine
Low spike	—	—	—	—	—	—	—	—
Middle spike	—	—	—	—	—	—	—	—
High spike	5.80	1.01	17.5	1.97	33.9	2.76	120	8
Methylpseudoephedrine
Low spike	—	—	—	—	—	—	—	—
Middle spike	—	—	—	—	—	—	—	—
High spike	6.37	0.319	5.00	3.81	59.8	4.94	104	8

Collaborators' Comments

Laboratory A could not detect any peaks in the plasma samples that corresponded to the ephedra alkaloids.

Laboratories A–C, E, and G noted that the urine samples seem to contain interferences which made identification and integration of the ephedra alkaloids difficult.

Laboratory I could not maintain a column temperature of 25°C with its column heater. To main tain constant temperature and ensure reproducible retention times, they increased the column temperature to 30°C. Resolution and selectivity of the method were not affected. The same laboratory noted that the tailing factor they observed for EP was 1.54, and the system suitability requirement for tailing factor of EP was not more than 1.5.

Laboratory C noted that the high buffer concentration in the mobile phase (100mM) damaged their UV flow cell and pump pistons after 1 month of continuous use. No other laboratories noted any adverse effects from the high buffer concentration.

Laboratories A, G, H and I noted that a precipitate formed when the SPE sample eluate was diluted with 500mM H₃PO₄. This precipitate was most likely ammonium phosphate. It is not certain why other laboratories did not experience this phenomenon, but the precipitation may be temperature dependant.

No additional comments were received.

Performance Characteristics of the Study

Insufficient data was available to calculate repeatability (RSD_r) and reproducibility (RSD_R) for NE and EP for any test samples. RSD_r and RSD_R could only be calculated for NP, PS, ME, and MP for the high spike urine samples, and for NP for the middle spike urine samples. RSD_R was very high for all cases, with HORRAT values >2.7. Average recoveries ranged from 99.1% for PS in the high spike urine sample to 600% for NP in the middle spike urine sample. Positive results were obtained for NE, NP, EP, and ME in the blank urine samples by at least 5 laboratories. Blank results of ten approached or even exceeded those of the spiked test samples for several analytes.

There was insufficient data for any of the ephedra alkaloids in the plasma samples to conduct statistical analysis. Most collaborating laboratories either could not detect any alkaloids in the spiked samples, or the detected alkaloids were well below the calibration range of the method.

All determination coefficients (R²) for the calibration curves exceed 0.997.

Recommendations

Because of the high variability in results among collaborative laboratories, the difficulty in detecting the ephedra alkaloids in plasma samples, and the presence of interfering peaks in the urine samples, this method is not recommended for adoption as Official First Action.