Objective:

2

To optimize the pre-column derivation high performance liquid chromatography (HPLC) content determination method of

D

-mannose and

D

-glucose as well as the content determination method of narinhenin in

Dendrobium officinale

and

D

.

huoshanense

and compare the contents of

D

-mannose

D

-glucose and narinhenin between

D

.

officinale

and

D

.

huoshanense

.

Method:

2

A pre-column derivation HPLC method modified by

Chinese Pharmacopoeia

(Ch.P) 2015 was used to simultaneously determine the contents of

D

-mannose and

D

-glucose

with acetonitrile-0.02 mol·L

-1

ammonium acetate solution as mobile phase for gradient elution. Kromasil 100-5 C

18

was performed with the wavelength set at 250 nm

and the flow rate was 1 mL·min

-1

; column temperature was 30 ℃. HPLC content determination of narinhenin was performed on Kromasil 100-5 C

18

with the acetonitrile-methanol-0.4% phosphoric acid solution as mobile phase for gradient elution

and the wavelength was set at 290 nm; the flow rate was 0.8 mL·min

-1

and column temperature was 40 ℃.

Result:

2

D

-mannose and

D

-glucose showed a good linear relationship within the range of 0.15-3.0 μg and 0.075-2.25 μg (

r

=0.999 9); and their average recoveries were 99.01% (RSD 2.1%) and 101.69% (RSD 2.0%) respectively. In addition

the other methodological researches such as repeatability and durability all met the requirements. The contents of

D

-mannose(

C

m

)

D

-glucose(

C

g

) and sum of them (

C

m

+

C

g

) were 12.75%-36.40%

2.93%-18.39% and 19.23%-54.58% in 43 batch of

D

.

officinale

. Almost all of the results except very few samples reached the

D

-mannose standard in Ch.P 2015

and the total content of

D

-mannose and

D

-glucose was also up to the total polysccharide standard in Ch.P. The correlation between content and origin was not significant. The contents of

D

-mannose(

C

m

)

D

-glucose(

C

g

) and sum of them (

C

m

+

C

g

) were 14.33%-29.47%

6.64%-15.20%

and 25.73%-44.37% in 12 batch of

D

.

huoshanense

. These contents and ratio of peak areas of

D

-mannose to

D

-glucose (

A

m

/

A

g

) were within the scope of

D

.

officinale

'

s;

in addition

their average contents were basically the same with those in

D

.

officinale

(about 33%). Next

naringenin showed a good linear relationship within the range of 0.020 8-0.832 0 μg (

r

=0.999 9)

and its average recovery was 101.96% (RSD 1.8%). The content of naringenin was 0.053 2-0.122 4 mg·g

-1

(average value of 0.081 0 mg·g

-1

) in 11 batch of

D

.

officinale

slightly higher than 0.040 3-0.090 0 mg·g

-1

(average value of 0.068 3 mg·g

-1

) in 7 batch of

D

.

huoshanense

. All of these results of narinfenin did not reach the content lower limit in Ch.P.

Conclusion:

2

The method used to determinate the content of

D

-mannose and

D

-glucose is reproducible

and their sum content is possible to substitute the total polysccaride determination (with higher errors) in

D

.

officinale;

monosaccharide content determination can be used for quantitative quality control of

D

.

huoshanense

. However

it could not distinguish

D

.

officinale

and

D

.

huoshanense

by determining the contents of polysccharide

D

-glucose

D

-mannose and narinhenin

and shall be combined with other specificity methods for further identification.