Hormones

Basically, OHpak SB-802.5 HQ is a GFC column. Here, the column was used for the analysis of adrenal cortical hormones by reversed phase mode with a mixture of water/acetonitrile as an eluent.

Sample : Steroides mixture
1. Aldosterone
2. Corticosterone
3. Deoxycorticosterone

Column       : Shodex OHpak SB-802.5 HQ (8.0mmID*300mm)
Eluent       : H2O/CH3CN=70/30
Flow rate    : 1.0mL/min
Detector     : UV(240nm)
Column temp. : 30deg-C

Basically, Asahipak GF-310 HQ is a GFC/GPC column. Here, the column was used for the analysis of adrenal cortical hormones by multimode with a mixture of water/acetonitrile as an eluent.

Sample : 50micro-L
1. Aldosterone 0.125mg/mL
2. Corticosterone 0.25mg/mL
3. Deoxycorticosterone 0.25mg/mL

Column       : Shodex Asahipak GF-310 HQ (7.5mmID*300mm)
Eluent       : H2O/CH3CN=55/45
Flow rate    : 0.5mL/min
Detector     : UV(240nm)
Column temp. : 30deg-C

Three kinds of steroid were separated using OHpak SB-802.5 HQ (a column for GFC separation).

Sample : 0.01% each, 20micro-L
1. Cortisone
2. Prednisolone
3. Testosterone

Column       : Shodex OHpak SB-802.5 HQ (8.0mmID*300mm)
Eluent       : H2O/CH3CN=60/40
Flow rate    : 1.0mL/min
Detector     : UV(240nm)
Column temp. : 70deg-C

This shows an example for the detection of androgen (male sex hormone), by an optical rotation detector (ORD) and ODSpak F-411 ( a column for reversed phase chromatography ). Since the peak is negative, 5-androstene-3beta, 17beta-diol is judged to be levorotatory. 5-Androstene-3beta, 17beta-diol is reacted with hydroxysteroid dehydrogenase, and then, 4-androstene-3, 7-dione and testosteronethe are generated. The generation of two components are conceived at 2 min. Since the two peaks are both positive, the components are judged to be dextrorotatory.

Sample :
1. 4-Androstene-3,17-dione
2. Testosterone
3. 5-Androstene-3beta,17beta-diol

Column       : Shodex ODSpak F-411 (4.6mmID*150mm)
Eluent       : CH3OH
Flow rate    : 1.0mL/min
Detector     : ORD
Column temp. : Room temp.

This shows the analysis of catecholamines by isocratic elution using a polymer-based reversed phase column, RSpak DE-613.

Sample : Catecholamines
1. Norepinephrine
2. Epinephrine
3. Dopamine (DA)

Column       : Shodex RSpak DE-613 (6.0mmID*150mm)
Eluent       : 0.05M KH2PO4 + 0.05% H3PO4 aq.
Flow rate    : 0.6mL/min
Detector     : UV(254nm)
Column temp. : 25deg-C

This shows an analysis of DOPA, which is a precursor, and other catecholamines by isocratic elution using a polymer-based weak cation exchange column, Asahipak ES-502C 7C.

Sample : Catecholamines
1. DOPA
2. Epinephrine
3. Norepinephrine
4. Dopamine

Column       : Shodex Asahipak ES-502C 7C (7.5mmID*100mm)
Eluent       : 20mM Sodium malonate buffer(pH6.0)+ 0.5M NaCl
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : 30deg-C

Catecholamines in urine were analyzed using Asahipak ES-502C 7C (a column for weak cation exchange chromatography).

Sample : Catecholamines in Urine
E. Adrenaline
N. Norepinephrine
D. Dopamine

Column           : Shodex Asahipak ES-502C 7C (7.5mmID*100mm)
Eluent           : 0.05M Succinic acid + 0.15M sodium succinate + 0.5M EDTA(pH6.7)
Reaction reagent : (A); 0.1M glycylglycine containing 0.1M tartaric acid + 
                        0.1M boric acid + 2mM zinc sulphate(pH6.5)
                   (B); 0.25M potassium borate buffer(pH8.4) 
                        containing 0.01%(w/v) hexacyanoferrate(III)
Flow rate        : (Eluent) 1.0mL/min, (Reaction reagent) 0.35mL/min each				   
Detector         : Fruorescence (Ex 350nm, Em 500nm)
Column temp.     : 30deg-C
Reaction temp.   : 90deg-C

T. Seki et al., The Chemical Sciety of Japan, (7) (1986) 1040

Catecholamines and salsolinol were analyzed using Asahipak ES-502C 7C (a column for weak cation exchange chromatography) and an electrochemical detector (ECD).

Sample : Catecholamines
E. Epinephrine
NE. Norepinephrine
DA. Dopamine
SA. Salsolinol

Column       : Shodex Asahipak ES-502C 7C (7.5mmID*100mm)
Eluent       : 25mM Boric acid buffer + 75mM Succinic acid + 20mM EDTA(pH8.0)
Flow rate    : 1.0mL/min
Detector     : ECD(700mV, VS, Ag/AgCl)
Column temp. : 40deg-C

Courtesy of Dr. Chiba, Nihon University

Catecholamines and metabolites were analyzed using Asahipak ODP-50 6D.

Sample : Catecholamines
NE: Norepinephrine
E: Epinephrine
DOPA: 3,4-Dihydroxyphenylalanine
DA: Dopamine
5HT: 5-Hydroxytryptamine
DOMA: 3,4-Dihydroxymandelic acid
MHPG: 3-Methoxy-4-hydroxyphenylglycol
VMA: Vanillylmandelic acid
DOPAC: 3,4-Dihydroxyphenylacetic acid
HVA: Homovanilic acid
5HIAA: 5-Hydroxyindole-3-acetic acid

 

Column       : Shodex Asahipak ODP-50 6D (6.0mmID*150mm)
Eluent       : (A); 0.1M H3PO4(pH2.0)
               (B); 0.1M H3PO4(pH2.0)/CH3CN=70/30
               Linear gradient:
               0min to 10min, (A)
               afetr 10min, 2.5%/min gradient of (B)
Flow rate    : 0.8mL/min
Detector     : UV(280nm)

Gibberellin homologues were analyzed using Asahipak ES-502N 7C (a column for weak anion exchange chromatography).

Sample : Gibberellin

Column       : Shodex Asahipak ES-502N 7C (7.5mmID*100mm)
Eluent       : Acetic acid/H2O/CH3OH=0.1/0.4/99.5
Flow rate    : 1.5mL/min
Detector     : UV(210nm)
Column temp. : 50deg-C

Courtesy of Dr. Yamaguchi, The Unversity of Tokyo

Gibberellin isomers were analyzed using Asahipak ES-502N 7C (a column for weak anion exchange chromatography).

Sample : Gibberellin

Column       : Shodex Asahipak ES-502N 7C (7.5mmID*100mm)
Eluent       : Acetic acid/H2O/CH3OH=0.1/0.4/99.5
Flow rate    : 1.5mL/min
Detector     : UV(210nm)
Column temp. : 50deg-C

Courtesy of Dr. Yamaguchi, The University of Tokyo

Recycle analysis of ecdyson from vitex fiherii was performed using Asahipak GS-320 20G ( a multimode column ).

Sample : Ecdysone

Column    : Shodex Asahipak GS-320 20G (20.0mmID*500mm)
Eluent    : CH3OH
Flow rate : 3mL/min

Courtesy of Dr. Kubo, University California, Berkley

Steroid hormones like estradiol are excreted in animals as aggregates – a form presenting no hormonal activity. It has however been reported that upon removal of water, they disassemble to release the free active form, which has prompted efforts to characterize such aggregates. In this application, estradiol and its metabolic by-products, i.e. 11 compounds including estrone and a variety of estroriol glucuronides were analyzed with the polymer-based NH2P40-2D column in HILIC mode with LC/MS/MS detection.

Sample : 0.1ppm each, 5micro-L
1. Estrone-3-sulfate
2. β-Estradiol-3-sulfate
3. Estriol-3-sulfate
4. Dehydroepiandrosterone-3-sulfate
5. Estrone-3-glucuronide
6. β-Estradiol-17-glucuronide
7. β-Estradiol-3-glucuronide
8. Estriol-3-glucuronide
9. Estriol-16-glucuronide
10. Dehydroepiandrosterone-3-glucuronide
11. Androsterone-3-glucuronide

Column　  : Shodex NH2P40-2D (2.0mmID*150mm)
Eluent　　: A; 10mM NH4OH H2O
            B; 10mM NH4OH CH3CN
Gradient  : linear gradient: 
            B%, 22% (0-5min), 22-70% (5-10min), 70% (10-15min)
Flow rate : 0.2mL/min
Detector  : ESI-MS/MS (Polarity : Negative）
Column oven : 40deg-C

Data courtesy of Mr. Atsushi Yamamoto, Osaka City Institute of Public Health and Environmental Sciences