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Analysis of gossypol by high performance liquid chromatography
Journal of Ethnopharmacology, 20 (1987) 1-11 Elsevier Scientific Publishers Ireland Ltd.
ANALYSIS OF GOSSYPOL CHROMATOGRAPHY
BY HIGH PERFORMANCE
LIQUID
MU-ZOU WANG Department of Analytical Chemistry, Institute of Materiu Medica, Sciences, Nan Wei Lu, Be&ng 100050 (China)
Chinese Academy
of Medical
(Accepted March 3, 1987)
Summary Several high performance liquid chromatographic methods for the analysis of gossypol in different kinds of sample were developed. (1) Pure (70 : 30 : 40) gossypol: separation on C,, column with MeOH/H,O/CHCl, containing 0.1% H,PO, as mobile phase or on SO,H column with MeOH/ citrate buffer (pH 6.3) (55:45) as mobile phase was recommended. With these systems, minute amounts of contaminants difficult to separate by other HPLC systems could be determined. (2) Plant material: acetone was selected as the extraction solvent. After evaporation of acetone from the extract, the residue was redissolved in 1% HOAc in CHCl,. An aliquot of this solution was chromatographed and quantified by peak area method. The mean recovery of pure gossypol added to plant material was 91.1? 1.1% (S.D.). (3) Plasma sample: a HPLC method with electrochemical detector was developed. A plasma sample with glutathione as protective agent and gossypol dimethyl ether as internal standard was introduced on to a C,, pre-column. By using column-switching technique, a certain part of the eluate containing gossypol and gossypol dimethyl ether was subjected to a C, analytical column for further separation. MeOH/citrate buffer (pH 3.2) (80:20) was used as the mobile phase. The optimum potential for detection was +0.6V vs. Ag-AgCl. The assay sensitivity was 5 nglml. This method is sensitive and selective, suitable for clinical pharmacokinetic studies.
Introduction Gossypol is a polyphenolic binaphthaldehyde compound originally isolated from the seeds of cotton, plants of the genus Gossypium. Since gossypol has been demonstrated to have an antifertility action in males and since it has also been found recently that gossypol may be used in the 037%8741/87/$04,20 @ 1987 Elsevier Published and Printrd in Ireland
Scientific Publishers
Ireland Ltd.
2
treatment of endometriosis and menopausal functional bleeding, much attention has been paid to it. A variety of methods have been developed to assay gossypol such as calorimetry (Pans, 1977), non-aqueous titration (Sha and Wang 1980), polarography (Jiang and Zhow, 19841, paper chromatography (Schramn and Benechict, 19581, thin-layer chromatography (Song, 1980) gas chromatography (McClure, 1971) etc. However, these methods suffer the disadvantage of low specificity, limited sensitivity or the requirement of prior derivatization. Recently, high performance liquid chromatography has been used for the analysis of gossypol (Abou-Donia et al., 1981; Nomeir and Abou-Donia, 1982; Marcelle et al., 1984). In order to meet the requirements for the systematic study of gossypol in our institute, studies on high performance liquid chromatographic methods for various purposes were carried out. (Wang et al., 1985a,b). Two new HPLC systems for qualitative and quantitative analysis of pure gossypol were developed first to meet the needs of quality control. Later, a new method for the quantitative determination of gossypol in plant material was developed. The latter method is suitable for the assay of crude material used for gossypol extraction and to determine the gossypol content in processed cotton-seed cake. Although the metabolism and physiological disposition of gossypol in animals have been studied using calorimetric or radioisotopic methods in different species of animals (Wang et al., 1979; Tan et al., 1980), no report on the clinical pharmacokinetics of gossypol has been found. One of the reasons is the lack of a sensitive and selective method to determine the very low concentration of gossypol in the plasma of volunteers. Now a HPLC method with electrochemical detector and column switching technique has been designed and developed. This method has been found to be quite satisfactory for this special purpose. Materials Chemical
and methods and plant resources
Gossypol, in the form of gossypol-acetic acid, and gossypol dimethyl ether (Fig. 1) were prepared in the Department of Organic Chemistry of this Institute. Chemically bounded phase for HPLC: YWG-C,,, -SO,H, -C,H,, -NH,, -CN (10 km, Tianjin Second Chemical Factory, China), Lichrosorb RP-2, RP-8, RAT (10 km, Merck, F.R.G.), Perisorb PA 6, a polyamide stationary phase (30-40 km, Merck), Nucleosil-C 18 (Macherey-Nagel, F.R.G.), reduced glutathione (Sigma, U.S.A.). All other chemicals and solvents were of analytical grade. Cotton plant was collected from the suburb of Beijing and identified at the Institute as Gossypium hirsutum L.
CH
/T
CH3
Cc3
CH3
‘CH,
Gossypol
U-0
A
CH3
OH
Fig. 1. Chemical
CHO
/%
CH3
Gossypol
Equipment
OH
CH3
CH3
dimethyl ether
structures.
resources
Schimadzu LC-4A high performance liquid chromatography with Chromatopac C-R2A.X data processor: the system for analysis of plasma samples was composed of two valves, two pumps, pre-column and analytical columns. Valve A was a Rheodyne 7125 injection valve, pump A was a constant flow high pressure pump (Shanghai Chemical Engineering School, China), Valve B and Pump B were accessories of Schimadzu LC-4A HPLC. A Pye Unicam PU-4022 electrochemical detector with a glassy carbon working electrode and a Ag-AgCl reference electrode was used for detec10 p_rn (30 x 4.0 mm I.D.), analytical column tion, pre-column Nucleosil-C,,, Lichrosorb-C,, 10 pm (250 x 4.0 mm I.D.). All columns were slurry packed under high pressure. Analytical
methods
Purity control of gossypol Approximately 5 mg of gossypol sample were accurately weighed into a 5-ml volumetric flask. A solution of 1% acetic acid in choloroform was added into the flask to dissolve the sample and then made to the mark. After mixing, a volume of 1 ml was pipetted out and diluted with the same solution to 10 ml. Four microlitres of this diluted solution were injected
4
into the liquid chromatograph. Analysis was performed by either one of the following HPLC systems. The quantity was determined by peak area method. (1) YWG-C,, column (250 x 4.0 mm), 0.1% phosphoric acid in MeOH/ H,O/CHCl, (70: 30:40) as mobile phase, 254 nm detection, flow rate 1 ml/ min. (2) YWG-SO,H column (250 x 4.0mm), MeOH/citrate buffer (pH 6.3) (55 : 45) as mobile phase, 254 nm detection, flow rate 1 ml/min. Determination
of gossypol
content
in plant material
A pulverized sample (100-300 mg) of stem, root, seed or seed cake of cotton plant was accurately weighed into a glass-stoppered test tube. Five millilitres of acetone were added to macerate for 16 h. The content was filtered under reduced pressure through filter paper into a 25-ml roundbottom flask. The test tube and the sample residue in the funnel were washed with two successive 3-ml portions of fresh acetone. The combined filtrates were evaporated to dryness under vacuum and the residue dissolved in 10.0 ml of 1% acetic acid in chloroform solution. A 4-~1 aliquot of this solution was injected and chromatographed under the same conditions as above. Determination of trace amounts of gossypol in human plasma Sample preparation and handling. To each 5-ml test tube were added
0.2 ml of 1% heparin solution and 0.1 ml of 0.2 M reduced glutathione solution and the mixture evaporated to dryness under vacuum. Volumes of l-2 ml human blood were placed in these tubes immediately after sampling, mixed thoroughly and stored in an iced Dewar flask (no more than 6 h). Before analysis, the sample was centrifuged for 15 min and the plasma transferred into another glass-stoppered test tube and kept at -40°C. The frozen plasma samples were thawed shortly before analysis and a volume of 0.2-0.4 ml was placed in 5-ml test tubes and 80 ng of the internal standard, gossypol dimethyl ether, and 0.6 ml of acetonitrile added. The tubes were vortexed and centrifuged. The supernatant was transferred into another conical glass tube containing 1 ml of 0.1 M citrate buffer solution (pH 3.2). After mixing and centrifuging for 10 min the protein-free samples were kept at 0°C ready for HPLC analysis. HPLC analysis. Chromatographic conditions: the mobile phase was MeOH/O.l M citrate buffer (pH 3.2) (80:20) with a flow rate of 1.5 and 1.2 ml/min for pump A and B, respectively. The electrochemical detector potential of the glassy carbon electrode was set at + 0.6 V vs. Ag-AgCl reference electrode. Assay procedure: the assay was performed using the column-switching technique. The procedure can be resolved into four steps (Fig. 2). (1)Injected 1 ml of protein-free sample into the loop of valve A, while
Valve A
Valve E3
Fig. 2. Procedures of column-switching
technique. See text for details.
pump A and I3 were eluting the C,, pre-column and C, analytical column, respectively. (2) when valve A was switched to “injection” position, the sample was driven into pre-column by pump A; this step lasted 1 min to let the polar contaminants be eluted out and discarded. (3) When valve B was switched to “injection” position, the eluent containing gossypol and internal standard were eluted from the precolumn into the analytical column. This step lasted for 4 min. (4) When valve B and A were switched back to “load” position again, the condition was actually the same as in step (I). Thus, the less polar contaminants could not get into the analytical column and were discarded. The pre-column may be cleaned by injecting 2 ml of methanol into the loop followed by normal elution as in step (2). Results and discussion HPLC method of gossypol After testing a number of stationary phases (-C,,, -C,, -C,, -SO,H, -NH,, -CN, -C,H, and Perisorb PA 6) and solvent systems, we found that -C,, and -SO,H columns gave better results than those used in previous reports for the separation of gossypol from its related contaminants.
6
On YWG-C,, column, the mobile phase used by Abou-Donia et al. (1981) was modified with addition of a certain amount of chloroform to form a system of MeOH/H,O/CHCl, (70: 30:40) containing 0.1% H,PO,. With this HPLC system, minute amounts of contaminants difficult to separate by other systems could be detected (Fig. 3). However, it is still difficult to make quantitative separation. It has been reported that gossypol is unstable in alcohol. New peaks may appear on the chromatogram of a methanol solution of gossypol after standing (Fig. 3). Though gossypol in methanol is only converted into a series of hemiketal and ketal forms not really “decomposed”, yet the appearance of other peaks will interfere with the determination of gossypol. The HPLC mobile phase used here contained methanol as a component, but it showed no serious influence on gossypol. Only one peak could be obtained in the case of pure gossypol under this chromatographic condition, as in this situation the separation was performed in a closed system and in a short period. After further comparison, we found that on YWG-SO,H column using MeOH/citrate buffer (55 : 45) as the mobile phase, more favourable results could be obtained. Among the pH range tested, pH 6.3 buffer gave the highest resolution (Fig. 41, under this condition the standard curve for gossypol acetic acid using the peak-area method was linear over the range of 0.1-0.8 pg. A similar mechanism for the separation with this HPLC system to that
L 0
5
IO
min
Fig. 3. HPLC of gossypol-HOAc: column, YWG-C,, (25 cm x 4.0 mm); mobile phase, 0.1% H,PO, in MeOH/H,O/CHCl, (70:30:40); flow rate, l.Oml/min; detection, 254 nm; range, 0.16 a.u.f.s. (1) Pure gossypol-HOAc; (2) Gossypol-HOAc in MeOH after 24 h. 1 gossypolHOAc peak, 2 decomposition peak. (3) Crude gossypol-HOAc 1,2 impurities.
,
‘4
a
12
min
Fig. 4. HPLC of gossypol-HOAc: column, YWG-SO,H citrate buffer (pH 6.3) (55:45); flow rate, 1.0 ml/min; a,b,c, impurities.
(25 cm x 4.0 mm); mobile phase, MeOH detection, 254 nm; range 0.16 a.u.f.s.
on a C,, phase was proposed. The phenyl group of YWG-SO,H bonded phase may have the same rule of the octadecyle group of C,, phase, while the strong acidic -SO,H group depressed the ionization of the phenolic hydroxyl groups of gossypol and resulted in a sharp peak. Other cation exchange bonded phases, such as Lichrosorb KAT, and -C,H, bonded phases have been tested, but they did not give acceptable results. A batch of gossypol acetic acid was recrystallized six times with acetic acid acetone. The corresponding contents after each recrystallization are as follows: starting crude material 83.6%; (1) 965%; (2) 98.9%; (3) 99.0%; (4) 99.5%; (5)101.2%; (6) 100.8%. Analysis
of plant material
The mixed solvent, used by Nomeir and from plant material, was dissolved in this standing for 15 min.
95% ethanol/water/ether/acetic acid (715 : 285: 220 : 0.2) Abou-Donia (1982) for extraction of gossypol was tried. However, we found that if gossypol solvent extra peaks would be revealed on This is due to the presence of alcohol. Therefore,
I3
some non-alcoholic solvent systems, 1% HOAc in CHCl,, acetone and 70% acetone, were compared. The results showed that the efficiency of extraction was in the order of 70% acetone > acetone > 1% HOAc in CHCl,. Because of the high absorbance of acetone at 254 nm, acetone must be evaporated off before HPLC analysis, and the residue redissolved in 1% HOAc in CHCl,. Although 70% acetone can complete the extraction in a much shorter time than pure acetone, it is more difficult to evaporate off. Therefore, we considered that acetone is a better extraction solvent as a whole. The mean recovery of pure gossypol added to plant samples was 97.1 ? 1.1% (SD.) (N = 6). Various parts of dry sample of gossypium hirsutum were analyzed and the gossypol contents were as follows: root 0.15%; stem 0.003%; cotton seeds 0.74%; cotton seed cake 0.097%. Determination
of gossypol
in human plasma
Gossypol is easily oxidized. In order to keep it unchanged before analysis, the effects of antioxidants and temperature on the stability of gossypol in human plasma samples were studied. Results showed that reduced glutathione exhibited the best protective effect. After adding reduced glutathione and keeping at 0°C for 6 h, the loss of gossypol in plasma was found to be less than 5.6%, while unprotected control samples suffered a loss of 80%. When stored at -3O”C, no appreciable loss was detected for at least 2 months. Gossypol is a polyphenolic hydroxy compound which exhibits electrochemical activity. Therefore, the electrochemical detector was chosen in the HPLC assay for its high sensitivity. The electrochemical behaviour of gossypol and gossypol dimethyl ether on a glassy carbon electrode was first studied to find out the lowest potential that gave rise to steady current signals. The hydrodynamic voltammograms were tested (Fig. 5). The applied voltages at which gossypol and gossypol dimethyl ether gave a plateau current were +0.6V and +O.SV, respectively. Because at +O.SV many interfering peaks were observed, + 0.6 V was chosen as the detector potential. The purification of a plasma sample by conventional solvent extraction gave only 60% recovery. A C,, pre-column enrichment procedure was chosen to retain and concentrate the gossypol in plasma. However, this led to the appearance of some large contaminant peaks, both polar and non-polar, which rendered the analysis difficult and caused bluntness of the electrode (Fig. 6). A column-switching technique was designed to solve this problem. This technique allowed entry of only a certain part of the eluate which contained the components for analysis from the pre-column into the analytical column. Other parts containing contaminants were discarded directly from the pre-column. A chromatogram was obtained in which the pre-gossypol contaminant peaks decreased markedly and the
_,’
0
/I
0.4
/ I
I
0.6
0.8
I
I
1.0 E ( Vvs. Ag/AgCl 1
Fig. 5. Hydrodynamic votammograms of gossypol (0)and gossypol dimethyl ether (x). non-polar interfering peaks disappeared (Fig. 7). This design may also be a very useful technique for the analysis of other complicated biochemical samples. The standard curve for gossypol, using peak height ratio method was linear over the range of 5-250 ng/ml, (r = 0.999). The minimum detectable concentration of gossypol in plasma is 5 ng/ml (signal/noise, 3: 1). The mean recovery of this method was 94.4 ? 6.4% (S.D.) (N = 9). The reproducibility of the method was tested by analyzing a blank human plasma sample spiked with 30.0 and 100.0 ng/ml of gossypol six times within 1 day. The results expressed as mean values and standard deviation were 26.5 ? 0.8 ng/ml and 100.5 t 2.4 ng/ml, respectively. A bulk plasma sample protected by adding glutathione and kept at -30°C was analyzed repeatedly over 15 days. The day-to-day variation was 69.3 jr 2.6 ng/ml. A single oral dose of 20 mg of gossypol was given to five male adults. Blood samples were taken and analyzed. The average level of gossypol was found to be 851-993 ng/ml after 4-6 h and 9.9-21.9 ng/ml after 192 h. These results showed that the blood concentration was in the determinable range and this method can serve as a standard method for clinical pharmacokinetic studies. post-gossypol
Separate
determination
of (+ I- and (-)-gossypol
Recently, the resolution of racemic gossypol was achieved and the antifertility activity of ( -+), (+ 1, (-1 isomers in male rats was studied.
10
15 C
!
‘uI
60.0 ‘20.0
10.0 min
Fig. 6. Chromatogram nique.
-r
-ii-
A-
60 1060
0
1050
min (I
of normal human plasma without
1
(II 1
using the column-switching
Fig. 7. Chromatogram of (I) human plasma containing gossypol and gossypol (IS.1 and (II) drug-free plasma using the colunm-switching technique.
tech-
dimethyl
ether
Preliminary results showed that (-I-gossypol given in half of the dose of the (2 > isomer produced antifertility activity comparable to that of the latter, while ( + )-gossypol was shown to be inactive. For further study of the activities of (+ )- and (-)-gossypol, it is necessary to develop an analytical method that can assay the isomers separately. A method treating gossypol with optically active amines followed by HPLC separation of the diastereometric amino compounds was tested. Many amines were studied and we found some of them formed pairs of diastereomers with different retention time and could be easily separated and determined by HPLC. The results will be published elsewhere and the study on the assay of ( + )- and (- I-gossypol in plasma sample is also under way. Acknowledgements The author gratefully thanks Professors Huang Liang, Zhou-tong and Lei-hai Peng for reviewing the manuscript.
Hui
References Abou-Donia, S.A., Lasker, J.M. and Abou-Donia M.B. (1981) High-performance liquid chromatographic analysis of gossypol. Journal of Chromatography 206, 606-610. Jiang, Y. and Zhou, T.-H. (1984) Polarographic study of gossypol. Actu Phurmuceutica Sinica 19, 195-201. Marcelle, G.R., Ahmed, M.S., Pezzuto, J.M., Cordell, G.A., Waller, D.P., Soejarto, D.D. and Fong, H.H.S. (19841 Analysis of gossypol and gossypol acetic acid by high performance liquid chromatography. Journal of Pharmaceutical Sciences 73, 396-398.
11 MC Clure, M.A. (1971)
Gas liquid chromatography
of gossypol. Journal
of Chromatography
54,
25-31. Nomeir, A.A. and Abou-Donia, M.B. (1982) Gossypol: high performance liquid chromatographic analysis and stability in various solvents. Journal of the American Oil Chemists’ Society 59, 546-599. Pons, W.A. (1977) Gossypol analysis: past and present. Journal of the Association of Official Analytical Chemists 60, 252-259. Schramn, G. and Benedict, J.H. (1985) Quantitative determination of traces of free gossypol in fats, oils and fatty acid by paper chromatography. Journal of the American Oil Chemists’ Society 35, 371-375. Sha, S.-Y. and Wang, Y.-P. (1980) Non-aqueous titration of gossypol and its preparations. Chinese Pharmaceutical Bulletin 15, 143-144. Song, Y.-W. (1980) Thin-layer chromatography of gossypol acetic acid. Yaojiangongzuo Tongxun 10, 67-71. Tan, X.-C., Zhu, M.-K. and Shi, Q.-X. (1980) Comparative studies on the absorption, distribution and excretion of “C-gossypol in four species of animals. Acta Pharmaceutics Sinica 15, 212-217. Wang, N.-G., Li, G.-X., Chen, Q.-Q. and Lei, H.-P. (1979) The metabolism of gossypol in viva. Chinese Medical Journal 59, 596-599. Wang, M.-Z., Wang, J.-S., Li, B.-L. and Gao, F.-Y. (1985a) Determination of gossypol with high performance liquid chromatography. Acta Pharmaceutics Sinica 20, 682-688. Wang, M.-Z., Wu, D.-F. and Yu, Y.-W. (1985b) High performance liquid chromatography with electrochemical detection of gossypol in human plasma. Journal of Chromatography, Biomedical Application 343, 387-396.
ANALYSIS OF GOSSYPOL CHROMATOGRAPHY
BY HIGH PERFORMANCE
LIQUID
MU-ZOU WANG Department of Analytical Chemistry, Institute of Materiu Medica, Sciences, Nan Wei Lu, Be&ng 100050 (China)
Chinese Academy
of Medical
(Accepted March 3, 1987)
Summary Several high performance liquid chromatographic methods for the analysis of gossypol in different kinds of sample were developed. (1) Pure (70 : 30 : 40) gossypol: separation on C,, column with MeOH/H,O/CHCl, containing 0.1% H,PO, as mobile phase or on SO,H column with MeOH/ citrate buffer (pH 6.3) (55:45) as mobile phase was recommended. With these systems, minute amounts of contaminants difficult to separate by other HPLC systems could be determined. (2) Plant material: acetone was selected as the extraction solvent. After evaporation of acetone from the extract, the residue was redissolved in 1% HOAc in CHCl,. An aliquot of this solution was chromatographed and quantified by peak area method. The mean recovery of pure gossypol added to plant material was 91.1? 1.1% (S.D.). (3) Plasma sample: a HPLC method with electrochemical detector was developed. A plasma sample with glutathione as protective agent and gossypol dimethyl ether as internal standard was introduced on to a C,, pre-column. By using column-switching technique, a certain part of the eluate containing gossypol and gossypol dimethyl ether was subjected to a C, analytical column for further separation. MeOH/citrate buffer (pH 3.2) (80:20) was used as the mobile phase. The optimum potential for detection was +0.6V vs. Ag-AgCl. The assay sensitivity was 5 nglml. This method is sensitive and selective, suitable for clinical pharmacokinetic studies.
Introduction Gossypol is a polyphenolic binaphthaldehyde compound originally isolated from the seeds of cotton, plants of the genus Gossypium. Since gossypol has been demonstrated to have an antifertility action in males and since it has also been found recently that gossypol may be used in the 037%8741/87/$04,20 @ 1987 Elsevier Published and Printrd in Ireland
Scientific Publishers
Ireland Ltd.
2
treatment of endometriosis and menopausal functional bleeding, much attention has been paid to it. A variety of methods have been developed to assay gossypol such as calorimetry (Pans, 1977), non-aqueous titration (Sha and Wang 1980), polarography (Jiang and Zhow, 19841, paper chromatography (Schramn and Benechict, 19581, thin-layer chromatography (Song, 1980) gas chromatography (McClure, 1971) etc. However, these methods suffer the disadvantage of low specificity, limited sensitivity or the requirement of prior derivatization. Recently, high performance liquid chromatography has been used for the analysis of gossypol (Abou-Donia et al., 1981; Nomeir and Abou-Donia, 1982; Marcelle et al., 1984). In order to meet the requirements for the systematic study of gossypol in our institute, studies on high performance liquid chromatographic methods for various purposes were carried out. (Wang et al., 1985a,b). Two new HPLC systems for qualitative and quantitative analysis of pure gossypol were developed first to meet the needs of quality control. Later, a new method for the quantitative determination of gossypol in plant material was developed. The latter method is suitable for the assay of crude material used for gossypol extraction and to determine the gossypol content in processed cotton-seed cake. Although the metabolism and physiological disposition of gossypol in animals have been studied using calorimetric or radioisotopic methods in different species of animals (Wang et al., 1979; Tan et al., 1980), no report on the clinical pharmacokinetics of gossypol has been found. One of the reasons is the lack of a sensitive and selective method to determine the very low concentration of gossypol in the plasma of volunteers. Now a HPLC method with electrochemical detector and column switching technique has been designed and developed. This method has been found to be quite satisfactory for this special purpose. Materials Chemical
and methods and plant resources
Gossypol, in the form of gossypol-acetic acid, and gossypol dimethyl ether (Fig. 1) were prepared in the Department of Organic Chemistry of this Institute. Chemically bounded phase for HPLC: YWG-C,,, -SO,H, -C,H,, -NH,, -CN (10 km, Tianjin Second Chemical Factory, China), Lichrosorb RP-2, RP-8, RAT (10 km, Merck, F.R.G.), Perisorb PA 6, a polyamide stationary phase (30-40 km, Merck), Nucleosil-C 18 (Macherey-Nagel, F.R.G.), reduced glutathione (Sigma, U.S.A.). All other chemicals and solvents were of analytical grade. Cotton plant was collected from the suburb of Beijing and identified at the Institute as Gossypium hirsutum L.
CH
/T
CH3
Cc3
CH3
‘CH,
Gossypol
U-0
A
CH3
OH
Fig. 1. Chemical
CHO
/%
CH3
Gossypol
Equipment
OH
CH3
CH3
dimethyl ether
structures.
resources
Schimadzu LC-4A high performance liquid chromatography with Chromatopac C-R2A.X data processor: the system for analysis of plasma samples was composed of two valves, two pumps, pre-column and analytical columns. Valve A was a Rheodyne 7125 injection valve, pump A was a constant flow high pressure pump (Shanghai Chemical Engineering School, China), Valve B and Pump B were accessories of Schimadzu LC-4A HPLC. A Pye Unicam PU-4022 electrochemical detector with a glassy carbon working electrode and a Ag-AgCl reference electrode was used for detec10 p_rn (30 x 4.0 mm I.D.), analytical column tion, pre-column Nucleosil-C,,, Lichrosorb-C,, 10 pm (250 x 4.0 mm I.D.). All columns were slurry packed under high pressure. Analytical
methods
Purity control of gossypol Approximately 5 mg of gossypol sample were accurately weighed into a 5-ml volumetric flask. A solution of 1% acetic acid in choloroform was added into the flask to dissolve the sample and then made to the mark. After mixing, a volume of 1 ml was pipetted out and diluted with the same solution to 10 ml. Four microlitres of this diluted solution were injected
4
into the liquid chromatograph. Analysis was performed by either one of the following HPLC systems. The quantity was determined by peak area method. (1) YWG-C,, column (250 x 4.0 mm), 0.1% phosphoric acid in MeOH/ H,O/CHCl, (70: 30:40) as mobile phase, 254 nm detection, flow rate 1 ml/ min. (2) YWG-SO,H column (250 x 4.0mm), MeOH/citrate buffer (pH 6.3) (55 : 45) as mobile phase, 254 nm detection, flow rate 1 ml/min. Determination
of gossypol
content
in plant material
A pulverized sample (100-300 mg) of stem, root, seed or seed cake of cotton plant was accurately weighed into a glass-stoppered test tube. Five millilitres of acetone were added to macerate for 16 h. The content was filtered under reduced pressure through filter paper into a 25-ml roundbottom flask. The test tube and the sample residue in the funnel were washed with two successive 3-ml portions of fresh acetone. The combined filtrates were evaporated to dryness under vacuum and the residue dissolved in 10.0 ml of 1% acetic acid in chloroform solution. A 4-~1 aliquot of this solution was injected and chromatographed under the same conditions as above. Determination of trace amounts of gossypol in human plasma Sample preparation and handling. To each 5-ml test tube were added
0.2 ml of 1% heparin solution and 0.1 ml of 0.2 M reduced glutathione solution and the mixture evaporated to dryness under vacuum. Volumes of l-2 ml human blood were placed in these tubes immediately after sampling, mixed thoroughly and stored in an iced Dewar flask (no more than 6 h). Before analysis, the sample was centrifuged for 15 min and the plasma transferred into another glass-stoppered test tube and kept at -40°C. The frozen plasma samples were thawed shortly before analysis and a volume of 0.2-0.4 ml was placed in 5-ml test tubes and 80 ng of the internal standard, gossypol dimethyl ether, and 0.6 ml of acetonitrile added. The tubes were vortexed and centrifuged. The supernatant was transferred into another conical glass tube containing 1 ml of 0.1 M citrate buffer solution (pH 3.2). After mixing and centrifuging for 10 min the protein-free samples were kept at 0°C ready for HPLC analysis. HPLC analysis. Chromatographic conditions: the mobile phase was MeOH/O.l M citrate buffer (pH 3.2) (80:20) with a flow rate of 1.5 and 1.2 ml/min for pump A and B, respectively. The electrochemical detector potential of the glassy carbon electrode was set at + 0.6 V vs. Ag-AgCl reference electrode. Assay procedure: the assay was performed using the column-switching technique. The procedure can be resolved into four steps (Fig. 2). (1)Injected 1 ml of protein-free sample into the loop of valve A, while
Valve A
Valve E3
Fig. 2. Procedures of column-switching
technique. See text for details.
pump A and I3 were eluting the C,, pre-column and C, analytical column, respectively. (2) when valve A was switched to “injection” position, the sample was driven into pre-column by pump A; this step lasted 1 min to let the polar contaminants be eluted out and discarded. (3) When valve B was switched to “injection” position, the eluent containing gossypol and internal standard were eluted from the precolumn into the analytical column. This step lasted for 4 min. (4) When valve B and A were switched back to “load” position again, the condition was actually the same as in step (I). Thus, the less polar contaminants could not get into the analytical column and were discarded. The pre-column may be cleaned by injecting 2 ml of methanol into the loop followed by normal elution as in step (2). Results and discussion HPLC method of gossypol After testing a number of stationary phases (-C,,, -C,, -C,, -SO,H, -NH,, -CN, -C,H, and Perisorb PA 6) and solvent systems, we found that -C,, and -SO,H columns gave better results than those used in previous reports for the separation of gossypol from its related contaminants.
6
On YWG-C,, column, the mobile phase used by Abou-Donia et al. (1981) was modified with addition of a certain amount of chloroform to form a system of MeOH/H,O/CHCl, (70: 30:40) containing 0.1% H,PO,. With this HPLC system, minute amounts of contaminants difficult to separate by other systems could be detected (Fig. 3). However, it is still difficult to make quantitative separation. It has been reported that gossypol is unstable in alcohol. New peaks may appear on the chromatogram of a methanol solution of gossypol after standing (Fig. 3). Though gossypol in methanol is only converted into a series of hemiketal and ketal forms not really “decomposed”, yet the appearance of other peaks will interfere with the determination of gossypol. The HPLC mobile phase used here contained methanol as a component, but it showed no serious influence on gossypol. Only one peak could be obtained in the case of pure gossypol under this chromatographic condition, as in this situation the separation was performed in a closed system and in a short period. After further comparison, we found that on YWG-SO,H column using MeOH/citrate buffer (55 : 45) as the mobile phase, more favourable results could be obtained. Among the pH range tested, pH 6.3 buffer gave the highest resolution (Fig. 41, under this condition the standard curve for gossypol acetic acid using the peak-area method was linear over the range of 0.1-0.8 pg. A similar mechanism for the separation with this HPLC system to that
L 0
5
IO
min
Fig. 3. HPLC of gossypol-HOAc: column, YWG-C,, (25 cm x 4.0 mm); mobile phase, 0.1% H,PO, in MeOH/H,O/CHCl, (70:30:40); flow rate, l.Oml/min; detection, 254 nm; range, 0.16 a.u.f.s. (1) Pure gossypol-HOAc; (2) Gossypol-HOAc in MeOH after 24 h. 1 gossypolHOAc peak, 2 decomposition peak. (3) Crude gossypol-HOAc 1,2 impurities.
,
‘4
a
12
min
Fig. 4. HPLC of gossypol-HOAc: column, YWG-SO,H citrate buffer (pH 6.3) (55:45); flow rate, 1.0 ml/min; a,b,c, impurities.
(25 cm x 4.0 mm); mobile phase, MeOH detection, 254 nm; range 0.16 a.u.f.s.
on a C,, phase was proposed. The phenyl group of YWG-SO,H bonded phase may have the same rule of the octadecyle group of C,, phase, while the strong acidic -SO,H group depressed the ionization of the phenolic hydroxyl groups of gossypol and resulted in a sharp peak. Other cation exchange bonded phases, such as Lichrosorb KAT, and -C,H, bonded phases have been tested, but they did not give acceptable results. A batch of gossypol acetic acid was recrystallized six times with acetic acid acetone. The corresponding contents after each recrystallization are as follows: starting crude material 83.6%; (1) 965%; (2) 98.9%; (3) 99.0%; (4) 99.5%; (5)101.2%; (6) 100.8%. Analysis
of plant material
The mixed solvent, used by Nomeir and from plant material, was dissolved in this standing for 15 min.
95% ethanol/water/ether/acetic acid (715 : 285: 220 : 0.2) Abou-Donia (1982) for extraction of gossypol was tried. However, we found that if gossypol solvent extra peaks would be revealed on This is due to the presence of alcohol. Therefore,
I3
some non-alcoholic solvent systems, 1% HOAc in CHCl,, acetone and 70% acetone, were compared. The results showed that the efficiency of extraction was in the order of 70% acetone > acetone > 1% HOAc in CHCl,. Because of the high absorbance of acetone at 254 nm, acetone must be evaporated off before HPLC analysis, and the residue redissolved in 1% HOAc in CHCl,. Although 70% acetone can complete the extraction in a much shorter time than pure acetone, it is more difficult to evaporate off. Therefore, we considered that acetone is a better extraction solvent as a whole. The mean recovery of pure gossypol added to plant samples was 97.1 ? 1.1% (SD.) (N = 6). Various parts of dry sample of gossypium hirsutum were analyzed and the gossypol contents were as follows: root 0.15%; stem 0.003%; cotton seeds 0.74%; cotton seed cake 0.097%. Determination
of gossypol
in human plasma
Gossypol is easily oxidized. In order to keep it unchanged before analysis, the effects of antioxidants and temperature on the stability of gossypol in human plasma samples were studied. Results showed that reduced glutathione exhibited the best protective effect. After adding reduced glutathione and keeping at 0°C for 6 h, the loss of gossypol in plasma was found to be less than 5.6%, while unprotected control samples suffered a loss of 80%. When stored at -3O”C, no appreciable loss was detected for at least 2 months. Gossypol is a polyphenolic hydroxy compound which exhibits electrochemical activity. Therefore, the electrochemical detector was chosen in the HPLC assay for its high sensitivity. The electrochemical behaviour of gossypol and gossypol dimethyl ether on a glassy carbon electrode was first studied to find out the lowest potential that gave rise to steady current signals. The hydrodynamic voltammograms were tested (Fig. 5). The applied voltages at which gossypol and gossypol dimethyl ether gave a plateau current were +0.6V and +O.SV, respectively. Because at +O.SV many interfering peaks were observed, + 0.6 V was chosen as the detector potential. The purification of a plasma sample by conventional solvent extraction gave only 60% recovery. A C,, pre-column enrichment procedure was chosen to retain and concentrate the gossypol in plasma. However, this led to the appearance of some large contaminant peaks, both polar and non-polar, which rendered the analysis difficult and caused bluntness of the electrode (Fig. 6). A column-switching technique was designed to solve this problem. This technique allowed entry of only a certain part of the eluate which contained the components for analysis from the pre-column into the analytical column. Other parts containing contaminants were discarded directly from the pre-column. A chromatogram was obtained in which the pre-gossypol contaminant peaks decreased markedly and the
_,’
0
/I
0.4
/ I
I
0.6
0.8
I
I
1.0 E ( Vvs. Ag/AgCl 1
Fig. 5. Hydrodynamic votammograms of gossypol (0)and gossypol dimethyl ether (x). non-polar interfering peaks disappeared (Fig. 7). This design may also be a very useful technique for the analysis of other complicated biochemical samples. The standard curve for gossypol, using peak height ratio method was linear over the range of 5-250 ng/ml, (r = 0.999). The minimum detectable concentration of gossypol in plasma is 5 ng/ml (signal/noise, 3: 1). The mean recovery of this method was 94.4 ? 6.4% (S.D.) (N = 9). The reproducibility of the method was tested by analyzing a blank human plasma sample spiked with 30.0 and 100.0 ng/ml of gossypol six times within 1 day. The results expressed as mean values and standard deviation were 26.5 ? 0.8 ng/ml and 100.5 t 2.4 ng/ml, respectively. A bulk plasma sample protected by adding glutathione and kept at -30°C was analyzed repeatedly over 15 days. The day-to-day variation was 69.3 jr 2.6 ng/ml. A single oral dose of 20 mg of gossypol was given to five male adults. Blood samples were taken and analyzed. The average level of gossypol was found to be 851-993 ng/ml after 4-6 h and 9.9-21.9 ng/ml after 192 h. These results showed that the blood concentration was in the determinable range and this method can serve as a standard method for clinical pharmacokinetic studies. post-gossypol
Separate
determination
of (+ I- and (-)-gossypol
Recently, the resolution of racemic gossypol was achieved and the antifertility activity of ( -+), (+ 1, (-1 isomers in male rats was studied.
10
15 C
!
‘uI
60.0 ‘20.0
10.0 min
Fig. 6. Chromatogram nique.
-r
-ii-
A-
60 1060
0
1050
min (I
of normal human plasma without
1
(II 1
using the column-switching
Fig. 7. Chromatogram of (I) human plasma containing gossypol and gossypol (IS.1 and (II) drug-free plasma using the colunm-switching technique.
tech-
dimethyl
ether
Preliminary results showed that (-I-gossypol given in half of the dose of the (2 > isomer produced antifertility activity comparable to that of the latter, while ( + )-gossypol was shown to be inactive. For further study of the activities of (+ )- and (-)-gossypol, it is necessary to develop an analytical method that can assay the isomers separately. A method treating gossypol with optically active amines followed by HPLC separation of the diastereometric amino compounds was tested. Many amines were studied and we found some of them formed pairs of diastereomers with different retention time and could be easily separated and determined by HPLC. The results will be published elsewhere and the study on the assay of ( + )- and (- I-gossypol in plasma sample is also under way. Acknowledgements The author gratefully thanks Professors Huang Liang, Zhou-tong and Lei-hai Peng for reviewing the manuscript.
Hui
References Abou-Donia, S.A., Lasker, J.M. and Abou-Donia M.B. (1981) High-performance liquid chromatographic analysis of gossypol. Journal of Chromatography 206, 606-610. Jiang, Y. and Zhou, T.-H. (1984) Polarographic study of gossypol. Actu Phurmuceutica Sinica 19, 195-201. Marcelle, G.R., Ahmed, M.S., Pezzuto, J.M., Cordell, G.A., Waller, D.P., Soejarto, D.D. and Fong, H.H.S. (19841 Analysis of gossypol and gossypol acetic acid by high performance liquid chromatography. Journal of Pharmaceutical Sciences 73, 396-398.
11 MC Clure, M.A. (1971)
Gas liquid chromatography
of gossypol. Journal
of Chromatography
54,
25-31. Nomeir, A.A. and Abou-Donia, M.B. (1982) Gossypol: high performance liquid chromatographic analysis and stability in various solvents. Journal of the American Oil Chemists’ Society 59, 546-599. Pons, W.A. (1977) Gossypol analysis: past and present. Journal of the Association of Official Analytical Chemists 60, 252-259. Schramn, G. and Benedict, J.H. (1985) Quantitative determination of traces of free gossypol in fats, oils and fatty acid by paper chromatography. Journal of the American Oil Chemists’ Society 35, 371-375. Sha, S.-Y. and Wang, Y.-P. (1980) Non-aqueous titration of gossypol and its preparations. Chinese Pharmaceutical Bulletin 15, 143-144. Song, Y.-W. (1980) Thin-layer chromatography of gossypol acetic acid. Yaojiangongzuo Tongxun 10, 67-71. Tan, X.-C., Zhu, M.-K. and Shi, Q.-X. (1980) Comparative studies on the absorption, distribution and excretion of “C-gossypol in four species of animals. Acta Pharmaceutics Sinica 15, 212-217. Wang, N.-G., Li, G.-X., Chen, Q.-Q. and Lei, H.-P. (1979) The metabolism of gossypol in viva. Chinese Medical Journal 59, 596-599. Wang, M.-Z., Wang, J.-S., Li, B.-L. and Gao, F.-Y. (1985a) Determination of gossypol with high performance liquid chromatography. Acta Pharmaceutics Sinica 20, 682-688. Wang, M.-Z., Wu, D.-F. and Yu, Y.-W. (1985b) High performance liquid chromatography with electrochemical detection of gossypol in human plasma. Journal of Chromatography, Biomedical Application 343, 387-396.