Submited on: 03 Jul 2013 08:57:14 AM GMT
Published on: 03 Jul 2013 10:40:00 AM GMT
 

  • What are the main claims of the paper and how important are they?

    This paper identifies a reverse phase chromatography method for finding the concentration of of a vitamin E called alpha-tocopherol. More specifically, the paper describes the use of HPLC in determining the amount of alpha-tocopherol in E. guineenis leaf extracts. They report the accuracy of the method with the percent recovery of the reference compounds at 96-105%. They also make claims on the precicison and limits of detection amounts for this compound. 

    The introduction tells how this compound might have been used when it was first discovered, but it is not clear on how determining the concentration of E. guineenis would be relavent in pharmacology. It would have been more helpful if the exact plans for use of this technique had been mentioned. 


  • Are these claims novel? If not, please specify papers that weaken the claims to the originality of this one.

    Yes this work is novel. However, there are some other methods out there. In the article, it was claimed that this new method's retention time is 6 minutes (which is true from the data given) compared to previous methods taking 25 minutes. 

    I then looked at their citations to find that one reference (Dauqan, E., et al., Vitamin E and Beta Carotene Composition in Four Different Vegetable Oils) reported four similar analysis times to be ‘less than 25 minutes’. Another of the references (Tasioula-Margari et al, Simultaneous determination of phenolic compounds and tocopherols in virgin olive oil using HPLC and UV detection) reported retention times of 10.3 and 21.2 minutes.  And the third reference (Hu, W., et al., Comparison of isopropanol and hexane for extraction of vitamin E and oryzanols from stabilized rice bran) gave a time of 15 minutes. From these values, I do not think it is accurate to state that current methods take 25 minutes in this report. These times also do not seen to correspond directly to the time that is being reported the in article (retention time), as some of the times stated refer to the total processing time for a sample. 


  • Are the claims properly placed in the context of the previous literature?

    For the most part. The article does build off some previous methods and some of these I just mentioned in answering the last question. Basically, this method looks to provide a better way of doing things and reduces the time it takes, though I do think that they should have been a little more truthful in the use of the timing of previous works, as mentioned in the last question. I thought it would have been helpful for the use of converting the compound to FAME to be mentioned more clearily. 


  • Do the results support the claims? If not, what other evidence is required?

    The results do support the claims but it would have been nice to see more of the results and the exact values that were obtained in the experiement. I tried to go back and verify the detection and quanitification limits, percent recovery, and the concentrations using the equations that were given in the methods. However, for each of the equations I could not find all values neceassry for the equation so I could not verify the calculations. Values that should of been included are things like the standard deviation of the y intercept and the amounts after the method was performed. 


  • If a protocol is provided, for example for a randomized controlled trial, are there any important deviations from it? If so, have the authors explained adequately why the deviations occurred?

    They seemed to follow their methods as described. 


  • Is the methodology valid? Does the paper offer enough details of its methodology that its experiments or its analyses could be reproduced?

    Yes, I believe that the process for the equipment used could be duplicated. It seems that all necessary values and steps are present. It might have been helpful to know the value of the 8 concentration points in the 0.2- 100 microg/mL range for the precision method.It was helpful that they included the equations they used. Though percent recovery is a pretty well known equation so it might not have been necessary. Limits of detection and quantification method equations were a good addition, though.


  • Would any other experiments or additional information improve the paper? How much better would the paper be if this extra work was done, and how difficult would such work be to do, or to provide?

    Additional information on the methods used and the amounts found in the lab would be useful. It would also be nice to see the data that gave them a linearized fit of R2=0.999. This should not be difficult information to include. 

    In the ‘quantification of alpha-tocopherol concentration in E. guineensis extracts’ section, it is unclear where they are injecting the extracts. The methods here should be a little more detailed but the design seems to be appropriate and there does not seem to be any fatal flaws. 


  • Is this paper outstanding in its discipline? (For example, would you like to see this work presented in a seminar at your hospital or university? Do you feel these results need to be incorporated in your next general lecture on the subject?) If yes, what makes it outstanding? If not, why not?

    I would not be interested in seeing this information at a seminar etc. It could prove to be a useful method but it is not something that cannot be lived without, as there are other methods of accomplishing this and it is just an analytical procedure. 


  • Other Comments:

    There seem to be multiple formatting and grammatical mistakes in this work. Some of these include:

    • In the introduction, the second sentence does not seem to be gramatically correct. 
    • Capitalize preparation in ‘Standard preparation’ title, since it is capitalized in the title above it.
    • In ‘limits of detection and quantification’ before the equation/at the end of the text introducing the equation, it should be a colon not a semi colon.
    • In ‘quantification of alpha-tocopherol concentration in E. guineensis extracts’, there should be not a period after the %w/w equatioTitle of section (results and discussion) shouldn’t be at the end of the page. 
    • Title of section (results and discussion) shouldn’t be at the end of the page.
    • Third paragraph of page 4, first sentence. Mean should be plural I believe.
    • Last sentence of paragraph 5 in results and discussion- suggested should be suggesting.  
  • Competing interests:
    .
  • Invited by the author to review this article? :
    No
  • Have you previously published on this or a similar topic?:
    No
  • References:
    None
  • Experience and credentials in the specific area of science:

    I study chemical engineering and am pursing a certificate in biopharmaceuticals. It is my 5th year. I am somewhat familiar with HPLC analysis and have some experience with publications in the medical field.

  • How to cite:  Beaven K .A validated RP-HPLC Method for Quantification of Alpha-tocopherol in Elaeis guineensis Leaf Extracts[Review of the article 'A validated RP-HPLC Method for Quantification of Alpha-tocopherol in Elaeis guineensis Leaf Extracts ' by Ismail Z].WebmedCentral 2014;5(12):WMCRW003169
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Review of
Posted by Ms. Ashley Arlinghaus on 07 Dec 2014 02:28:22 AM GMT Reviewed by Interested Peers

  • What are the main claims of the paper and how important are they?

    his paper claims that reverse-phase HPLC (RP-HPLC) is an appropriate process for quantifying the concentration of alpha-tocopherol present in leaves of E. guineensis. It also claims that conversion to fatty acid methyl esters (FAMEs) is more efficient for extracting alpha-tocopherol. Alpha-tocopherol is a type of vitamin E, which, like other compounds that fall under the vitamin E category, exhibits lipid solubility and antioxidant properties. Apparently, the fruit from E. guineensis yields large quantities of alpha-tocopherol, but, as the paper highlights, the field currently lacks a simple process for establishing the amount of alpha-tocopherol contained in the leaves. 

    Considering people do not normally consume the leaves of this plant, it is not readily apparent how quantification of the vitamin present in the leaves could be considered important. However, when one considers the current market for vitamin supplements, etc., it becomes evident that the leaves could be a viable source for a natural or organic vitamin supplements depending on the amount of alpha-tocopherol generally found in the leaves. Therefore, a simple method for quantifying alpha-tocopherol in E. guineensis could be advantageous for companies interested in using this plant to provide alpha-tocopherol for large-scale production of vitamin supplements. 


  • Are these claims novel? If not, please specify papers that weaken the claims to the originality of this one.

    The claims in this paper are novel regarding the use of E. guineensis leaves as the experimental source of alpha-tocopherol and the conversion of alpha-tocopherol from the leaf extracts to fatty acid methyl esters (FAMEs) before performing RP-HPLC. It is important to note that other papers have documented the use of RP-HPLC to identify and quantify alpha-tocopherol in sources other than E. guineensis leaves, such as cereal grains (Liu, S. et al. [2013]. Rapid determination of alpha-tocopherol in cereal grains using dispersive liquid-liquid microextraction followed by HPLC. J Sep Sci, 36[6]), cow’s milk (Ubaldi, A. et al. [2005]. Quick HPLC method to determine Vitamin E concentration in cow’s milk. Ann Fac Medic Vet di Pharma 25), and human serum (Abahusain, M. A. et al. [1998]. Determination of retinol, alpha-tocopherol, alpha- and beta-carotene by direct extraction of human serum using high performance liquid chromatography. Biomed Chromatogr, 12[2]). All three examples validated their RP-HPLC analysis methods based on accuracy, precision, linearity, and limits of detection and quantification just as this paper did. Considering this paper references Ubaldi et al, it appears that the authors of this paper are aware that other studies using RP-HPLC to detect and quantify alpha-tocopherol exist. 


  • Are the claims properly placed in the context of the previous literature?

    Yes, the claims are properly placed int he context of previous literature. This paper refers to several other publications to show how this study improved upon previous studies. It provides adequate references to highlight the drawbacks of saponification in order to support the claim that conversion to FAMEs is more efficient for extracting alpha-tocopherol than saponification. It also provides adequate references to show that the use of E. guineensis leaves is a deviation from prior sources of alpha-tocopherol, and therefore, a different approach for determining the amount of alpha-tocopherol present in the new source was necessary.


  • Do the results support the claims? If not, what other evidence is required?

    Yes, the results support the claims, but some additional data would make the claims more credible, most notably with regard to the determination of accuracy. In this paper, only three samples were used to determine percent recovery using the external standard addition method. This small sample size has a greater potential for variability and error. It would be better to increase the number of samples to 5 or 10 samples. Clarification with regard to the conversion of alpha-tocopherol to FAMEs would also help strengthen the claims. There is no statement indicating that the alpha-tocopherol standard was converted to FAMEs during its preparation. The differences in molecular properties could cause discrepancies in elution time or absorbance, which would then invalidate the results. 


  • If a protocol is provided, for example for a randomized controlled trial, are there any important deviations from it? If so, have the authors explained adequately why the deviations occurred?

    There appear to be no deviations from the protocol.


  • Is the methodology valid? Does the paper offer enough details of its methodology that its experiments or its analyses could be reproduced?

    The methodology regarding the use of the HPLC is valid. The explanation for converting the alpha-tocopherol from the leaf extracts to FAMEs is sound, but it is unclear whether only the leaf extracts were subject to this conversion or both the leaf extracts and the alpha-tocopherol standard were converted to FAMEs. If the standard was not converted to FAMEs, then it cannot be considered an appropriate standard for this experiment. Otherwise, the methods section provides enough detail for possible replication. 


  • Would any other experiments or additional information improve the paper? How much better would the paper be if this extra work was done, and how difficult would such work be to do, or to provide?

    The paper could be improved by the addition of several features:

    1. The graph of the calibration curve (linear regression) used to determine the linearity equation and R2 value. Having a visual representation of this data along with the chart presented would emphasize and validate how well the regression fits the data.
    2. Tables display values and averages for peak area that were used to determine accuracy and precision. The addition of these values would confirm that the math is correct and logical.
    3. Larger sample size for percent recovery as used to determine accuracy. A sample size larger than 3 (perhaps between 5 and 10) with additional calculations of the mean and standard deviation of the different values for percent recovery would strengthen the claim of accuracy.
    4. Indicate whether the LOD and LOQ were determined mathematically, experimentally, or both. If determined experimentally, indicate the sample sizes and concentration ranges that were used. If determined mathematically only, run tests at the LOD and LOQ and show that the mathematic prediction was correct. Doing so would justify the practicality of establishing LOD and LOQ.

    Only suggestion 3 would require significant extra work, and none of the additions should be difficult to provide.


  • Is this paper outstanding in its discipline? (For example, would you like to see this work presented in a seminar at your hospital or university? Do you feel these results need to be incorporated in your next general lecture on the subject?) If yes, what makes it outstanding? If not, why not?

    I would not consider this paper outstanding for its discipline, but that does not mean it is worthless. The data, as presented, do suggest that the logic behind this research is sound. However, some adjustments (listed in my response to the previous question) could be made in order to strengthen the authors’ claims. If such adjustment were made, I would likely reconsider my verdict on the paper’s standing within its discipline. 


  • Other Comments:

    None.

  • Invited by the author to review this article? :
    No
  • Have you previously published on this or a similar topic?:
    No
  • References:

    .

  • Experience and credentials in the specific area of science:

    .

  • How to cite:  Arlinghaus A .Review of [Review of the article 'A validated RP-HPLC Method for Quantification of Alpha-tocopherol in Elaeis guineensis Leaf Extracts ' by Ismail Z].WebmedCentral 2014;5(12):WMCRW003154
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