Development of a New LC-MS/MS Screening Method for Detection of 120 NPS and 43 Drugs in Blood

Round 1

Reviewer 1 Report

This manuscript described a screening method for the simultaneous detection of 163 substances (120 NPS and 43 BDZ) in blood samples by the dynamic multiple reaction monitoring mode based on LC-MS/MS. The analytical system selected in the manuscript is interesting, the developed LC-MS/MS analytical method is practical， and the experimental design is comprehensive (the authors assessed sample treatment, selectivity, specificity, LOD, LOQ, linearity, accuracy, inter-day precision, spike recovery, matrix effect, and stability). However, the innovation of this manuscript is not fully and clearly described, and some data needs to be added to the manuscript to support the relevant conclusions. Therefore, I recommend this manuscript for publication in Separations after revision:

1. Page 2, Lines 55-56: “This great interest has led to the development and validation of a great number of new multi-analyte LC-MS/MS screening methods mainly focused on NPS [6, 20-26]”.
   Compared with the reported "a great number of new multi-analyte LC-MS/MS screening methods", what are the advantages of the analytical method developed by the authors? Are there any disadvantages? The authors need to give a comparative conclusion in this regard to reflect the innovation of the paper.

2. There is only one paragraph in the entire Introduction. It is recommended to start a new paragraph from "The aim …" of Page 2, Line 56.

3. Page 3, Line 130: The authors start the gradient elution from 99% of the water phase. Even at the beginning, will the mobile phase with such a high water ratio have a negative effect on the column?

4. Figure 1: The time label on the abscissa is unclear, which is prone to ambiguity.

5. Figure 1: The authors need to adjust the font size in the picture to be larger, and the resolution of the picture needs to be adjusted to 300 dpi or higher.
6. Lines 258-259: It is recommended that the coefficient of determination for each substance should be given in Table 2.

7. Lines 291-293: It is recommended to give out chromatograms and quantitative data of real samples.

8. Line 56: “multi-analyte” should be corrected to “multi-analytes”.
9. Line 122: “MS Analysis” should be corrected to “MS analysis”.
10. Line 123: “an Agilent 6460 Triple Quad LC/MS” should be corrected to “an Agilent 6460 Triple Quad MS”.
11. Line 124: “(Agilent Technologies)”. The format here should be consistent with other similar formats (manufacturer, city, country).
12. Line 131: What does "within7 5 min" mean?

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

This is a well written and interesting paper. There are minor grammatical and English errors that should be corrected throughout, but they do not detract from the paper.

This paper describes an LC-MS/MS method to quantify 120 new psychoactive substances, in addition to benzodiazepines, opioids and additional drugs from whole blood. The method can chromatographically resolve most compounds and detection and validation was good for most QC levels and most compounds. 

It is not the most novel research, as it simply builds on previous research from the group (2016), altering the chromatography but using the same mobile phases, column and MS conditions. However, this is common in both analytical chemistry and toxicology and incremental progress is made on well established methods. It is an important addition to the literature, as it expands the number of drugs that can be screened in a single analytical run with minimal sample prep.

Introduction:

p2. ln73: sensibility should be sensitivity

Materials and methods:

Section 2.3: the wording of the gradient is confusing. Is it possible to word in such a way that the total run time is more apparent. i.e. "from 0-7.5 min, linear ramp from 0-5%B, from 7.5-12 min, ramp to 10%B, isocratic hold from 12 to 15 min...etc. It will allow the reader to better understand the elution characteristics of the compounds.

Table 1: Is it possible to list the drug class for each compound (benzo, opioid, SSRI, etc.) and group them together? It might make it easier for a reader to really focus on the drugs they are interested in and can highlight the chemical similarities and differences within and between classes.

Section 2.4.1: why were the chosen drugs used as interferences? Could you describe the selection? Are they common? Found at high concentrations when used, etc.

Table 2: Beside or below each parameter heading could you specify N replicates for each (i.e. accuracy (n=5)). In this way the table will truly stand alone as a reference for method validation.

Conclusion: you mention HRMS and it's usefulness in expanding screening for drugs, especially in the face of new and emerging substances. Given the quantitative capabilities of orbitraps and QToFs, could you comment on the place of a more targeted analytical platform like LC-MS/MS?

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

The article described a new LC-MS/MS screening method for the detection of 120 NPS and 43 drugs in blood samples, in which the LC-MS/MS was extended for the detection of forensic toxicological interest (i.e., benzodiazepines and other antidepressants). Overall, the experiments were carefully designed and well performed, and the manuscript was well written. The article should attract much attention from scientists working in forensic toxicology and analytical chemistry!

some minor points are presented below:

1) for the introduction, it's better to put it into two or three paragraphs for readability purpose;

2) Please check out through the references and make sure the style of the references is uniform. For instance, in references 6, 7 and 20, the first letters of all the words of the article name were capitalized, while some of the other references only the first letter of the first word was capitalized.

Author Response

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Author Response File: Author Response.pdf

Reviewer 4 Report

Ref.:  Manuscript ID: separations-1447303

Development of a new LC-MS/MS screening method for the detection of 120 NPS and 43 drugs in blood.

The article describes an analytical method for the quantitative analysis of 120 NPS and 43 classic drugs synthetic in blood. The method was validated and applied to 51 authentic cases (containing classic drugs). The methodology is scientifically sound (PP and LC-MS/MS), and it has a clear and useful application in forensic toxicology laboratories. The NPS crisis makes it necessary to have analytical tools for the detection of different types of these drugs in blood.

However, several issues should be addressed. Please, see below the specific comments.

Abstract: define MDPV.

-           Introduction: The authors should compare the present method with other similar published methods, indicate any limitation of those methodologies, and what is novel about the one presented.

-           The authors may also discus which are the most prevalent NPS in Italy (ex. literature).

-           Sample pre-treatment/validation: Blood origin: How many different type/sources of blood were investigated in the matrix effect experiment and authentic samples?  Is it always whole blood or do you analyse also plasma with the present method?

-           Validation/results: The authors should compare the concentrations (LLOQ and ranges) validated with the ones reported in previous recent publications. The LLOQ should be also validated in terms of variation (RSD%) and not only S/N.

- How the quality control (QC) were prepared? Did you check the quantification (at least for the classic drugs) with external QCs or proficiency test with the present method?

-           The fact that some analogues drugs did not fulfil the criteria should be discussed more in depth (ex. chromatographic baseline separation for those which present almost the same retention time and transitions, include specific figures chromatograms for those compounds, solutions). If they include these compounds  also in the same calibration curves, the quantification would not be neither OK (as there would be overlapping).

Table 2: Include the QC concentrations evaluated (in the legend or in the table).

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 4 Report

the manuscript can be published in the present form