Fast Analysis of Terpene Lactones in

Ginkgo Biloba Extract Using the

Agilent 1290 Infinity LC and Agilent
Poroshell 120 SB-C18 2.7 m Column

Application Note

Authors Abstract
Nick Severin The use of the Agilent 1290 Infinity LC coupled with the Agilent Poroshell 120
Jos. A. Schwabe SB-C18 3 100 mm, 2.7 m column can not only reduce solvent consumption, but
Green Bay, WI allows for faster run times. This is illustrated by comparing analyses of terpene
lactones in ginkgo biloba extract using the parameters outlined in the United States
Pharmacopeia (USP) and the 1290 Infinity LC with the Poroshell 120 SB-C18 column.
Introduction Table 1. Calibration Curves

Ginkgo biloba extract is derived from the leaves and seeds of Standard used USP #1291559 Lot # 10K042
the ginkgo tree, one of the oldest living tree species in the Sample Range R2
world. The herb is used to improve memory, treat depression, Bilobalide 32.776524.43 g/mL 1.00000
and maintain healthy blood circulation. Terpene lactones in
Ginkgolide J 10.671170.74 g/mL 0.99994
the extract inhibit platelet activating factors to improve blood
Ginkgolide C 12.506200.10 g/mL 0.99985
circulation. The extracts are standardized to contain approxi-
mately 6% terpene lactones. Ginkgo biloba is regulated as a Ginkgolide A 19.527312-424 g/mL 1.00000
dietary supplement, not a drug. Ginkgolide B 11.855189.686 g/mL 0.99997
All curves used quadratic equations
Ginkgo Biloba extraction conditions
Gingo Biloba extract source
Schwabe Extracta GmbH, Germany, Lot 2900270. Results and Discussion
Procedure The chromatogram in Figure 1 shows a time reduction 4 times
A 120-mg extract of sample was sonicated in 10 mL of a phos- less than the USP conditions, shown in Figure 2, and an 8-fold
phate buffer. The sample solution was transferred to a chro- solvent savings. The USP assay uses a 250 4.6 mm, 5.0 m
matographic tube filled with siliceous earth (Merck, Extrelut C-18 column. This compares to an Agilent Poroshell 120
NT20 column). To aid in the transfer, two additional 5-mL por- SB-C18, 100 3 mm, 2.7 m column using the 1290 Infinity
tions of the phosphate buffer were used. Allow the sample LC. Both systems use the same mobile phase, detector set-
solution to be absorbed by the column contents, about tings (Agilent 385 Evaporative Light Scattering Detector
15 minutes, before eluting with 100 mL of ethyl acetate. The (ELSD)), and temperature. The flow rate using the Poroshell
ethyl acetate elutant was evaporated under vacuum at 50 C. column was half the flow rate of the USP method.
The residue was dissolved in 20 mL of 50/50 methanol/water
solution.

Phosphate buffer
A 1.19-g amount of dibasic sodium phosphate and 8.25 g of
monobasic potassium phosphate were dissolved in 1,000 mL
of water and pH adjusted to 5.8.
Norm.

6.116 Ginkgolide A
Bilobalide
140

4.255 Ginkgolide C

6.429 Ginkgolide B
3.359
120

3.967 Ginkgolide J
100
80 Area: 171.692
60

6.855

7.735
7.348

7.950
7.555
7.123
40

5.272
3.713
3.010
2.552
2.099

20
0
1 2 3 4 5 6 7 8 min

Instrument parameters PMT gain 2.0

Temperature 30 C Data rate 40 Hz
Injection amount 3.0 L Flow rate 0.50 mL/min
ELSD detection conditions Mobile phase A Water
Instrument Agilent 385-ELSD Evaporative Mobile phase B Methanol
Light Scattering Detector,
Gradient Time (min) %A %B
model #4261A
0 75 25
Nebulizer temperature 40 C 7.5 52 48
Evaporator temperature 40 C 9.5 10 90
10.0 10 90
Nitrogen gas flow rate 1.50 SLM
10.5 75 25
12.5 75 25

Figure 1. Terpene lactones in ginkgo biloba extract. Agilent 1290 Infinity LC with an Agilent Poroshell 120 SB-C18 2.7 m column.

Norm.
11.762 Bilobalide

20.092 Ginkgolide A

175
21.176 Ginkgolide B
14.135 Ginkgolide C

28.016
28.371 28.516
26.213

150
23.193
23.734
13.150 Ginkgolide J

125
26.620

28.814
20.435 20.680

24.278

100
19.461

26.906

29.378

75
25.897
22.234

27.575
24.912
24.505
22.331
21.585
19.291
17.955
18.366

50
14.928
12.177
10.219
10.825

25
0
5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 min

Instrument parameters PMT gain 2.0

Temperature 30 C Data rate 40 Hz
Injection amount 15 L Flow rate 0.50 mL/min
Flow rate 1.0 mL/min Mobile phase A Water
ELSD detection conditions Mobile phase B Methanol
Instrument Agilent 385-ELSD Evaporative Gradient Time (min) %A %B
Light Scattering Detector, model 0 75 25
#4261A 23 52 48
28 52 48
Nebulizer temperature 40 C
30 25 75
Evaporator temperature 40 C 35 10 90
Nitrogen gas flow rate 1.50 SLM 40 75 25
50 75 25

Figure 2. Terpene lactones in ginkgo biloba extract. USP method using Phenomenex Luna C-18 250 4.6 mm, 5.0 m column.

3
Norm.

6.125 Ginkgolide A
3.362 Bilobalide
250

4.254 Ginkgolide C

6.438 Ginkgolide B
3.970 Ginkgolide J
200

150

100

50

0
1 2 3 4 5 6 7 8 min

Figure 3. Terpene lactone reference standard chromatogram (Agilent Poroshell method).

Standard used: USP #1291559 Lot # 10K042.
20.105 Ginkgolide A

Norm.
11.780 Bilobalide

21.190 Ginkgolide B
14.146 Ginkgolide C

120
13.145 Ginkgolide J

26.814
100

26.401 26.582
80
22.787 22.896

60
27.183

28.735
29.020
25.065
23.792
19.682

40
14.968

20
0
5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 min

Figure 4. Terpene lactone reference standard chromatogram (USP method).

Standard used: USP #1291559 Lot # 10K042.

Conclusions
In analyzing complex botanical extracts, the Agilent 1290
Infinity LC in conjunction with Agilent Poroshell 120 SB-C18
columns can speed up analysis time, saving cost in labor and
solvent use without any loss in resolution.

Reference
www.agilent.com/chem
Ginkgo Extract Monograph, United States Pharmacopeia,
USP36, NF31 p.1606. Agilent shall not be liable for errors contained herein or for incidental or consequential
damages in connection with the furnishing, performance, or use of this material.

For More Information Information, descriptions, and specifications in this publication are subject to change
without notice.

These data represent typical results. For more information on Agilent Technologies, Inc., 2014
our products and services, visit our Web site at Printed in the USA
January 23, 2014
www.agilent.com/chem. 5991-3856EN