Direct nano-electrospray ionization tandem mass spectrometry for the quantification and identification of metronidazole in its dosage form and human urine

A rapid, sensitive and direct nano-electrospray ionization-tandem mass spectrometry (NS-ESI-MS/MS) method, using an offline nanospray (NS) capillary, has been developed and validated for the analysis of metronidazole (MTZ). A mixture of 2 µl MTZ sample solution prepared in an ionization solvent consisting of methanol : water : formic acid in a ratio of 80 : 20 : 0.3, together with 2 µl of an internal standard (IS), 1,3,6-polytyrosine, is loaded into the back of the NS capillary. The NS capillary was fitted into the ion source at a distance of 3 mm between the NS tip and MS orifice. The sample is then analysed and acquired a sustainable signal that allowed for data compilation across various data points for MTZ identification and quantification. The quantification relied on the ratio of the [M + H]+ peaks of MTZ and IS with m/z values of 172.0717 and 182.0812, respectively, while the identification relied on the MS/MS of the precursor ions [M + H]+ of both compounds and their fragments at 128.05 for MTZ and 165.1 and 136.07 for the IS. The NS-ESI-MS/MS method was accurate and precise for the quantification of MTZ over the concentration range from 2.5 to 25 000 ng ml−1. The applicability of the method was confirmed by MTZ analysis in its pharmaceutical dosage form and detection of the analyte in clinical human urine samples without any sample treatment procedure.


Recommendation?
Accept with minor revision (please list in comments)

Comments to the Author(s)
The manuscript "Direct nano-electrospray ionization tandem mass spectrometry for quantification and identification of metronidazole in its dosage form and human urine" provides a well-established methodology protocols for ease of quantification and detection of MTZ in urine sample (especially in a clinical setting). The paper is well suited in the Royal Society scope and I recommend it to be published with minor grammatical corrections:

Are the interpretations and conclusions justified by the results? Yes
Is the language acceptable? Yes

Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? No

Recommendation?
Accept with minor revision (please list in comments)

Comments to the Author(s)
In this work, direct nano-electrospray ionization-tandem mass spectrometry (NS-ESI-MS/MS) was developed and validated to quantify MTZ in tablet dosage forms. Direct NS-ESI-MS/MS was also designed for continuous detection of drug intake of patients via screening of the drug in urine samples despite the presence of matrix interfering compounds or other co-administered drugs. Obviously, direct MS is of great importance due to combining the benefits of the detection and quantification without the separation technique in coupling with MS. Both of the originality and experimental data are good. In general term, the present study has enough quality to be published in Royal Society Open Science but some minor revisions are necessary before it may be accepted.

2-Standard Solutions and calibration
According to the results in Table 1 and linearity, the working standard solution range should be from 0.25-2500 µg/mL and subsequently, the calibration standard solutions ranging from 2.5 to 25000 ng/mL 3-The identity of parts a, b and c in figure 5 should be indicated.
4-Annotation of fragments should be corrected (underscore of molecule numbers) (H2O and NH3) 5-Detection of MTZ in human urine 5.1. "MTZ is also believed to reduce the desire for alcohol in alcoholism"; "Several LC-MS methods can easily study the presence of MTZ in blood or in urine but these cannot be carried out without sample pretreatment procedures"…References should be given 7-In section 3.3, the last sentence" The goal of calibration was to calibrate the instrument to detect the correct mass" is not needed 8-In section 3.5, the last two sentences should be deleted and instead should state that the same method for dosage method was applied. Title: Direct nano-electrospray ionization tandem mass spectrometry for quantification and identification of metronidazole in its dosage form and human urine Manuscript ID: RSOS-191336 Thank you for submitting the above manuscript to Royal Society Open Science. On behalf of the Editors and the Royal Society of Chemistry, I am pleased to inform you that your manuscript will be accepted for publication in Royal Society Open Science subject to minor revision in accordance with the referee suggestions. Please find the reviewers' comments at the end of this email.
The reviewers and handling editors have recommended publication, but also suggest some minor revisions to your manuscript. Therefore, I invite you to respond to the comments and revise your manuscript.
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Once again, thank you for submitting your manuscript to Royal Society Open Science. The chemistry content of Royal Society Open Science is published in collaboration with the Royal Society of Chemistry. I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Comments to the Author(s) In this work, direct nano-electrospray ionization-tandem mass spectrometry (NS-ESI-MS/MS) was developed and validated to quantify MTZ in tablet dosage forms. Direct NS-ESI-MS/MS was also designed for continuous detection of drug intake of patients via screening of the drug in urine samples despite the presence of matrix interfering compounds or other co-administered drugs. Obviously, direct MS is of great importance due to combining the benefits of the detection and quantification without the separation technique in coupling with MS. Both of the originality and experimental data are good. In general term, the present study has enough quality to be published in Royal Society Open Science but some minor revisions are necessary before it may be accepted.

2-Standard Solutions and calibration
According to the results in Table 1 and linearity, the working standard solution range should be from 0.25-2500 µg/mL and subsequently, the calibration standard solutions ranging from 2.5 to 25000 ng/mL 3-The identity of parts a, b and c in figure 5 should be indicated.
4-Annotation of fragments should be corrected (underscore of molecule numbers) (H2O and NH3) 5-Detection of MTZ in human urine 5.1. "MTZ is also believed to reduce the desire for alcohol in alcoholism"; "Several LC-MS methods can easily study the presence of MTZ in blood or in urine but these cannot be carried out without sample pretreatment procedures"…References should be given 7-In section 3.3, the last sentence" The goal of calibration was to calibrate the instrument to detect the correct mass" is not needed 8-In section 3.5, the last two sentences should be deleted and instead should state that the same method for dosage method was applied. 1-Trichomoniasis vaginalis should be italicized (p1, line 46) 1-Trichomoniasis vaginalis was italicized in the revised text.

2-Standard Solutions and calibration
According to the results in Table 1 and linearity, the working standard solution range should be from 0.25-2500 µg/mL and subsequently, the calibration standard solutions ranging from 2.5 to 25000 ng/mL 2-Concentrations were corrected in the text to match the results of Table 1. 5-Detection of MTZ in human urine 5.1. "MTZ is also believed to reduce the desire for alcohol in alcoholism"; "Several LC-MS methods can easily study the presence of MTZ in blood or in urine but these cannot be carried out without sample pretreatment procedures"…References should be given 5.1. References [48-50] were added in the text.

6-Data Extraction and Interpretation
There should be a singular formatting style when recalling figures. When referring to figure 4, parts a and b should appear in the text.