DRAFT 2: Major Project

            Hron et al. (1999) constructed a study to determine the amount of (+)-, (-)- and total gossypol in cottonseed using high performance liquid chromatography (HPLC) technique. Gossypol is the major active compound that causes temporary infertility in males. Gossypol in sufficient quantity can be toxic to animals. Bond connecting with the two naphthyl rings lead to steric hindrance between the functional groups of the molecule explaining the availability of both (+)- and (-)-isomers. Thus, scientists attempted to separate these two isomers. In Hron et al. (1999) study, acid-delinted cottonseed samples were selected to investigate in this study. The samples were ground and cracked, and then meats were separated out. The samples of meats were accurately weighed and derivatized by (R)-(-)-2-amino-1-propanol. A quantitative HPLC procedure was developed for the separation of (+)- and (-)-gossypol contained in cottonseed. An octadodecylsilane was used as reverse-stationary phase and eluted with acetonitrile: 20%potassium hydrogen phosphate (pH 3.0) (ratio 8:2) at 1.0 milliliter/minute. The (+)- and (-)-gossypol-2-amino-1-propanol complexes were separated at 1.4 and 2.6 minutes, respectively. The results showed that a crop of Upland cottonseed had (+)-enantiomer more than (-)-enantiomer, 67.6% and 32.4%, respectively; whereas, a seed sample from a Pima crop in Texas showed a higher amount of (-)-gossypol than (+)-gossypol, 53.1% and 46.9%, respectively. In conclusion, the HPLC method can distinguish gossypol from gossypol-like compound and quantify them while the official American oil chemist societies (AOCS) method can only detect total amount of gossypol. The researchers suggested that their new HPLC method can conveniently and quantitatively determine the isomers of gossypol in cottonseed. Moreover, they assumed that this HPLC method might possibly be scaled-up to provide materials for antiviral activity testing.

            This study provides scientific evidence to support herbal medicine in term of the quantitative analysis of each enantiomers of gossypol in medicinal plants which is necessary for herbal drug development. However, there are some limitations.

1)      The plant authentication should be carefully investigated. According to Mukherjee (2005), the specimen of plant should be compared with herbarium reference voucher specimens, or authenticated by a botanist to avoid mistakes over plant identification.

2)      This study had an objective to quantify the amount of active compound in medicinal plant. In experimental part, the researcher only described about the topic of linearity, accuracy and precision. ICH guideline (2005) summarized that the method validation was good practice for quantitative analysis. It was ensured that the analytical methodology was valid and reliable. Thus, the researchers should completely do in method validation for the most suitable method in quantifying the gossypol isomers.

3)      The researchers did not explain the method of AOCS in the part of experimental procedures. They did not use the statistical parameters to determine the differences of the result among the two methods; therefore, the researchers might have bias on data conclusion in explaining how this HPLC method was more suitable than AOCS method. They should have used pair t-test for determining the fitting of data.

The strength of this study is that HPLC technique is broadly accepted for many laboratories because HPLC has high reproducibility, high resolution, and can be detected in part per million levels. Moreover, another advantage of this research study is the clearly results that included text, tables and figures which were not required lengthy and complex explanation.

References

Hron, R., Kim, H., Calhoun, M., & Fisher, G. (1999). Determination of (+)-, (−)-, and Total Gossypol in Cottonseed by High-Performance Liquid Chromatography. Journal Of American Oil Chemist's Society, 76(11), 1351 - 1355.

Mukhejee, P.K. (2005). Quality control of herbal drug: an approach to evaluation of botanicals.
            New Delhi: Galaxy Printers.

International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Uses. (2005). Validation of Analytical Procedures: Text and Methodology Q2(R1). (pp. 1-13). Geneva: ICH.

DRAFT 1: Major Project

     Hron et al. (1999) constructed a study to determine the amount of (+)-, (-)- and total gossypol in cottonseed using high performance liquid chromatography (HPLC) technique. Gossypol is the major active compound that causes temporary infertility in males. Gossypol in sufficient quantity can be toxic to animals. Bond connecting with the two naphthyl rings lead to steric hindrance between the functional groups of the molecule explaining the availability of both (+)- and (-)-isomers. Thus, scientists attempted to separate these two isomers. In Hron et al. (1999) study, acid-delinted cottonseed samples were selected to investigate in this study. The samples were ground and cracked, and then meats were separated out. The samples of meats were accurately weighed and derivatized by (R)-(-)-2-amino-1-propanol. A quantitative HPLC procedure was developed for the separation of (+)- and (-)-gossypol contained in cottonseed. A octadodecylsilane was used as reverse-stationary phase and eluted with acetonitrile: 20%potassium hydrogen phosphate (pH 3.0) (ratio 8:2) at 1.0 milliliter/minute. The (+)- and (-)-gossypol-2-amino-1-propanol complexes were separated at 1.4 and 2.6 minutes, respectively. The results showed thata crop of Upland cottonseed had (+)-enantiomer more than (-)-enantiomer, 67.6% and 32.4%, respectively; whereas, a seed sample from a Pima crop in Texas showed a higher amount of (-)-gossypol than (+)-gossypol, 53.1% and 46.9%, respectively. In conclusion, the HPLC method can distinguish gossypol from gossypol-like compound and quantify them while the official American oil chemist societies (AOCS) method can only detect total amount of gossypol. The researchers suggested that their new HPLC method can conveniently and quantitatively determine the isomers of gossypol in cottonseed. Moreover, they assumed that this HPLC method might possibly be scaled-up to provide materials for antiviral activity testing.

            This study provides scientific evidence to support herbal medicine in term of the quantitative analysis of each enantiomers of gossypol in medicinal plants which is necessary for herbal drug development. However, there are some limitations.

1)      The plant authentication should be carefully investigated. According to Mukherjee (2005), the specimen of plant should be compared with herbariumreference voucher specimen, or authenticated by a botanist to avoid mistakes over plant identification.

2)      This study had an objective to quantify the amount of active compound in medicinal plant. In experimental part, the researcher only described about the topic of linearity, accuracy and precision. ICH guideline (2005) summarized that the method validation was good practice for quantitative analysis. It was ensured that the analytical methodology was valid and reliable. Thus, the researchers should completely do in method validation for the most suitable method in quantifying the gossypol isomers.

3)      The researchers did not explain the method of AOCS in the part of experimental procedures. They did not use the statistical parameters to determine the differences of the result among the two methods; therefore, the researchers might have bias on data conclusion in explaining how this HPLC method was more suitable than AOCS method. They should have used pair t-test for determining the fitting of data.

The strength of this study is that HPLC technique is broadly accepted for many laboratories because HPLC has high reproducibility, high resolution, and can be detected in part per million levels. Moreover, another advantage of this research study is the clearly results that included text, tables and figures which were not required lengthy and complex explanation.