Modern Approaches to Preparation of Body Fluids for Determination of Bioactive Compounds

Round 1

Reviewer 1 Report

The article presents an adequate review of the preparation of body fluid samples using miniaturized techniques.
In general, the article contains interesting and well-structured information, although it would be necessary to make some retouches for its improvement.
line 28: why has saliva not been included as a fluid of interest? Increasingly, this matrix is being researched as an alternative due to the non-invasive nature of its sampling, as well as the speed of response (think of drug tests for drivers).
Line 32: perhaps I would add a fourth factor: analyte stability. The sample matrix itself and the bioactive nature of the analytes can modify the concentration sought during sample treatment.
Section 2.1, solid phase extractions. I think there are two sample treatment techniques that should be included in the study: Stir Bar Sorptive Extraction (SBSE) and In Tube Solid Phase Microextraction (IT-SPME). Both are mature techniques, with dozens of applications for these types of samples, which would increase the scope of the article.
Section 3. Applications. In some cases, the information previously described in section 2 is repeated. The authors have two options: either merge both sections and, together with the brief description of the technique, including the applications, or they revise this section by eliminating the information already described and focusing only on the particularities of the selected procedures, wherever they exist.
Line 247: although the original authors include 96 samples in 2 minutes as a success, the subsequent analytical determination (LC-MS) is a bottleneck. These methodologies make sense if subsequent detection is performed by direct measurements, for example using colorimetric sensors or fluorescence measurements in the same wells where the sample is processed.
line 295: I would rewrite the phrase as "... was checked using human serum samples spiked with five statins ..."
Bibliography: only 4 references are from 2018, one from 2017 and one from 2016: only six from thirty-six. A review article should include a more up-to-date selection of bibliography.
Table 1: some superscripts _a _b appear in the text. It appears to be a formatting error that needs to be corrected.

Author Response

Respond for the Reviewer
We would like to thank very much the Reviewer for his valuable remarks and comments. We believe that after our corrections made according to the Reviewer’s directions, the current version of the manuscript has been considerable improved.
All our changes made in the manuscript were marked in green color.
Reviewer 1
Line 28: of course, saliva/oral fluid is an important alternative material for determination of drugs and especially drugs of abuse or narcotics in drivers. Its superior to routinely tested materials (blood and urine) mainly results from non-invasive nature of its sampling. However, please notice that in the statement: ‘Especially, plasma/serum, whole blood and urine constitute biological materials, which are examined routinely’ we have tried to express conventional character of the investigated biological materials. However, according to the Reviewer's suggestion, we rewrote the above mentioned sentence (into: ‘Plasma/serum, whole blood, urine and oral fluid constitute biological materials of special interest’), and we also include an example of micro-extraction technique application for this material.
Line 32: ‘chemical nature of analyte and type of sample matrix’ were added as a factor influencing on the choice of sample preparation method.
Section 2.1: according to the suggestion of the Reviewer, two extraction techniques: SBSE and IT-SPME were included in this section, as well as appropriate applications of these microextraction techniques were provided (section 3, line 250, and Table 1).
Section 3: this section was corrected according to the Reviewer suggestion and appropriate changes in text were marked in green color.
Line 247: we agree with the Reviewer’s comment that quickness of the developed sample treatment method should be matched with throughput of the whole developed analytical method. However, considering low concentrations of the determined drugs in blood samples, we think that it is rather problematic to find selective and sensitive enough detection method suitable for direct measurement of these analytes in the studied biological material. In any case, the finding out of such direct detection method(s) might be a challenge task for further investigations in this area.
Line 295 (at present line 352): the sentence was rewritten according to the Reviewer’s suggestion.
Bibliography: During review of publications for our article we tried to select up-to-date papers, however more interesting and representative (in our opinion) works we found mostly in the earlier period of time . However, finally, we added a number of the most current papers (especially review articles and papers concerning new materials used as sorbents, from 2018 and 2019) which were marked in green.
Table 1: we apologize for the fact that in the original version of our manuscript we did not provide appropriate legends for Tables 1 and 2. In the present (corrected version) we added the legends, and we believe that they will clarify the given superscripts.

Reviewer 2 Report

The review entitled: “Modern approaches to preparation of body fluids for 2 determination of bioactive compounds” deals with the modern techniques applied to the sample preparation of biological fluids for the analysis of bioactive compounds.

The manuscript is well written and organized, but I think something needs to be added/changed.

QUERY: what do the authors mean by bioactive compounds?

Generally, the term “bioactive compounds” is used for phytochemical compounds. It is appropriate for caffeine and theophylline, but most of the reported molecules in the manuscript are drugs. I recommend changing this term with “drugs”.

In the section 2.1 Solid phase extractions, please add a brief description of SPE.

Author Response

Respond for the Reviewer
We would like to thank the Reviewer for his remarks and comments. We believe that our explanations will satisfy the Reviewer.
Reviewer 2
What do the authors mean by bioactive compounds?
According to our knowledge, in literature, the term ‘bioactive compounds’ is used for the compounds which influence on biological functions of organism. Therefore, it may cover such compounds as medicines, drugs of abuse, narcotics or hormones.
Section 2.1: Although the fundamental principles of SPE are practically the same as for their derivative solid phase extraction techniques (considered in our review article), we intentionally did not include this source technique, because it is commonly known, as well as cannot be included in group of microextraction techniques.

Reviewer 3 Report

This article reviews the main microextraction techniques used for the analysis of biosamples. Although this article may compete with an article published in Separations (doi: 10.3390/separations4040036), a closer reading shows that both articles can be complementary. The review is concise (this is really welcome in a review) but gives a good overview of the approaches. However, this reviewer suggests some modifications to the manuscript in order to make its impact higher and to make it different enough from the previous commented article published in Separations.

1. References for further reading.

As it was previously indicated, the review is concise. This aspect will improve the engagement of the readers but some recent reviews articles (where each mentioned technique is presented in detail) must be introduced in the references. Some examples of potential reviews are:

· Single-drop microextraction (doi: 10.1016/j.trac.2018.09.016)

· Dispersive micro-solid phase extraction (doi: 10.1016/j.trac.2018.12.005)

· Micro solid-phase extraction (pipette tip and spin column) and thin film solid-phase microextraction: Miniaturized concepts for chromatographic analysis (doi: 10.1016/j.trac.2019.06.036)

2. Improving the coverage of the techniques.

The article can be improved if some updated techniques and approaches are at lest mentioned using general references.

a) Thin film microextraction must be mentioned in the SPME section as a derived technique.

b) One of the key trends in SPME is its coupling with MS to develop faster analytical methods. Please mention this trend. You can support this mention with a recent review article “Solid Phase Microextraction-mass spectrometry: Metanoia” (doi: 10.1016/j.trac.2018.12.030)

c) Electromembrane extraction is a powerful technique in the bioanalytical context. A reference to this technique is necessary.

d) The stirring- integrated techniques like stir bar sorptive extraction (SBSE) are not mentioned. They are powerful techniques in this context. (doi: 10.3390/separations4010006)

3. Improving the coverage of the materials

The development of new materials is also a key trend in microextraction that can be exploited in bioanalysis. There are many review articles published in MDPI that can be used to support this statement. For example, interesting materials are Fabric Phases (10.3390/separations5030040), ionic liquids (10.3390/molecules23061437) or MOFs (10.3390/separations6030033).

There are also some other materials that have been recently described for microextraction in bioanalysis like:

graphene oxide tablets (10.3390/molecules24071191)

paper (10.3390/molecules23061252 and 10.1007/s00216-014-8392-5)

bract (10.1016/j.jchromb.2019.04.037)

Cork (10.1016/j.jchromb.2018.10.021)

4. Other minor comments

· Please, do not use acronyms in the headings

· Line 72. Correct the author name.

· Use MNPs as the acronym for magnetic nanoparticles.

Author Response

Respond for the Reviewer
We would like to thank very much the Reviewer for his valuable remarks and comments. We believe that after our corrections made according to the Reviewer’s directions, the current version of the manuscript has been considerable improved.
All our changes made in the manuscript were marked in green color.
Reviewer 3
1. References for further reading
The three suggested review articles were included in our manuscript (section 2.2.1. – [29], section 2.1.4. – [22] and section 2.1.3 – [19])
2. Improving the coverage of the techniques
a. thin film microextraction was mentioned as a derived technique in section 2.1.1. Solid phase micrextraction and derived techniques (lines 80-86);
b. the trend consisting in coupling SPME with MS was mentioned and supported by the suggested review article (‘Solid phase micrextraction-mass spectrometry: Metanoia’), cited as [12] – lines 69-71;
c. electromembrane extraction as the extended concept of HF-LPME was indicated, and the appropriate review article [34] including its applications in pharmaceutical analysis was cited (lines 210-215).
d. The stir bar sorptive extraction (SBSE) technique was added and described in the separate section 2.1.5 (lines 141-155), and appropriate example applications of this technique were included in Table 1. The suggested review article was also added (cited as [25]).
3. Improving the coverage of the materials
The suggested review articles, as well as papers focusing on particular materials, recently described as sorbents in microextractions, were mentioned in Introduction (lines 46-49) and cited as [3-9].
4. Other minor comments
- The acronyms in the headlines in sections 2.1 and 2.2 were substituted by full names of the extraction techniques.
- Line 72 (at present line 90): the author name was corrected.
- The acronym ‘MNPs’ was used uniformly for magnetic nanoparticles (lines 129-130, line 132).

Round 2

Reviewer 1 Report

Paper has been improved over the first version, both in new techniques and in kind of samples.

In my opinion, now only small corrections are needed in the presented version.

CONTENT:

Lines 186-190: when DLLEME-SFO is presented, I would add a reference for this variant of the technique.

Page 19, Table 2. A superscript f) appears in the legend of the table, but I have not been able to find it in the text. Please check that it has not been omitted, or delete it from the legend.

Some English corrections:

Line 47: change “is introducing” by “the introduction”

Line 98: change “to reused” by “to reuse”

Line 118: change “amount” by “number” and “high cost” by “higher cost”

Line 150: change “to employed” by “to employ”

Line 154: change “emphasis their” by “emphasis in their”

Line 174: change “the creating” by “the creation”

Line 175: change “in the recent” by “in a recent”

Line 222: a subscript for the 2 in PPy-CH2-COOH formula is missing.

Line 297: change “god” by “better”

Line 308: change “can use as” by “can be used as”

Line 457: change “to quantitative” by “to quantify”

Line 533: reference 22, change “Cárdanes” by “Cárdenas” (see ref. 25)

Author Response

Response for Reviewer 1

Once again, we thank the Reviewer for a thorough review of our manuscript and for her/his comments. We have revised the manuscript in accordance with her/his suggestions. 

The changes in the manuscript have been made using the "Track Changes" function in Microsoft Word. 

On behalf of the authors

Katarzyna Madej, PhD

Reviewer 3 Report

The authors have considered my suggestions in the revision. Therefore I can recommend now  the publication of the article.

Author Response

Response for Reviewer 3

Once again, we thank the Reviewer for a thorough review of our manuscript and for her/his comments.

On behalf of the authors

Katarzyna Madej, PhD