Simultaneous Quantification of Propylthiouracil and Its N-β-d Glucuronide by HPLC-MS/MS: Application to a Metabolic Study

Round 1

Reviewer 1 Report

General comment: In this paper the authors developed and validated a HPLC-MS/MS method for the simultaneous quantification of PTU, which is a medication commonly prescribed for the management of hyperthyroidism and thyrotoxicosis, whose the mechanism of action is not fully understood, and for the quantification of a PTU metabolite, i.e. PTU  N-β-D glucuronide (PTU- 17 GLU). Before publication on Pharmaceuticals, I suggest major revisions.

 

Section 2.1 The authors should provide details about the MS conditions (spray voltage, capillary voltage, capillary temperature etc..)

Figure 1: Please, provide the title of x and y axis.

L90-91: “Developed with” instead of “developed by with”.

m/z should be in italics. Please correct through all the manuscript.

Figure 2: Please, provide the title of x and y axis and verify for all the figures of the manuscript.

Section 2.2 The authors should specify the guidelines that they followed in order to validate their method.

L158-L159. It’s not possible to talk about absolute recoveries, since they were expressed as percentage. As the values found ranged between 98.2% to 114%, it could be possible to demonstrate the absence of matrix effect. To this regard, see the following research article: “Analytical and bioanalytical chemistry,410(5), 1561-1569.”

 

L174-L176 “Compared to previously published bioanalytic approaches for propylthiouracil, our validated HPLC-MS/MS method filled the gap of quantifying glucuronide and was successfully applied to in vitro metabolism study” The authors should cite the references. Moreover, other studies are available in literature about the validation of method for the quantification of glucuronide in several biological matrices (for example plasma). Here, the authors do not report where the developed method could be applied. Pooled human liver microsomes? Please, argue about and clarify this.

 

 I suggest the authors to check for typing errors through the whole manuscript.

Author Response

Replies to Reviewer 1
We highly appreciate the important comments from the reviewer. We have amended the manuscript according to the comments. Here are the point-to-point replies:

General comment: In this paper the authors developed and validated a HPLC-MS/MS method for the simultaneous quantification of PTU, which is a medication commonly prescribed for the management of hyperthyroidism and thyrotoxicosis, whose the mechanism of action is not fully understood, and for the quantification of a PTU metabolite, i.e. PTU N-β-D glucuronide (PTU- 17 GLU). Before publication on Pharmaceuticals, I suggest major revisions.

Section 2.1 The authors should provide details about the MS conditions (spray voltage, capillary voltage, capillary temperature etc..)

We have supplied the information in the revised manuscript (lines 301 – 305):
The heat block temperature was set at 400℃ with the desolvation line (DL) temperature at 250℃ and interface temperature at 300℃. Other operating parameters were set as follows: conversion voltage, 10 kV; detector voltage, 2.08 kV; collision induced dissociation (CID) gas, 270 kPa; interface voltage, 4.0 kV; nebulizer gas, 2.0 L/min; heating gas, 10 L/min; and drying gas, 10 L/min.

Figure 1: Please, provide the title of x and y axis.

We have amended the manuscript as suggested (page 4).

L90-91: “Developed with” instead of “developed by with”.
We have amended the manuscript as suggested.
m/z should be in italics. Please correct through all the manuscript.

We have amended the manuscript and use italics as suggested.

Figure 2: Please, provide the title of x and y axis and verify for all the figures of the manuscript.

We have amended the manuscript as suggested (page 5).

Section 2.2 The authors should specify the guidelines that they followed in order to validate their method.

We have amended the manuscript as suggested (lines 341 - 345):
This bioanalytical method was validated with accordance to to the guidelines from FDA and International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines [30,31].

L158-L159. It’s not possible to talk about absolute recoveries, since they were expressed as percentage. As the values found ranged between 98.2% to 114%, it could be possible to demonstrate the absence of matrix effect. To this regard, see the following research article: “Analytical and bioanalytical chemistry,410(5), 1561-1569.”

We have amended the manuscript as suggested (lines 172 - 173) and added the article suggested by the reviewer as a reference [35].

L174-L176 “Compared to previously published bioanalytic approaches for propylthiouracil, our validated HPLC-MS/MS method filled the gap of quantifying glucuronide and was successfully applied to in vitro metabolism study” The authors should cite the references. Moreover, other studies are available in literature about the validation of method for the quantification of glucuronide in several biological matrices (for example plasma). Here, the authors do not report where the developed method could be applied. Pooled human liver microsomes? Please, argue about and clarify this.

We have amended the manuscript as suggested (lines 193 - 198)
The analytical methods for the quantification of PTU in biological matrices such as plasma, milk, urine and tissue samples have been reported before [23,25-27]. However, to the authors’ knowledge, there is no edstablised protocol suitable for the examination of glucuronidation of PTU. Therefore, in the present study, we developed and validated a reliable method for the simultaneous measurement of PTU and PTU-Glu and subsequently applied it to examine the in vitro metabolism of PTU in in HLMs.

I suggest the authors to check for typing errors through the whole manuscript.

The corresponding authors have double-checked the manuscript before returning it to the editorial office.

Reviewer 2 Report

The manuscript submitted by Li et al. is devoted to development and validation of an LC-MS/MS based method of quantitation propylthiouracil and its main metabolite glucuronide. This is the first method to determine both the compounds.

Having read the manuscript, I found that the experiment is very well planned and carried out. All data support the conclusions.

I have a couple of recommendations mentioned below.

1) From my point of view, using molar concentrations such as mM, μM etc. is more inconvenient for analysts who deal routine analyses. I would advise using mass concentrations (ng/mL. mcg/ml etc.) throughout the manuscript. Nevertheless, I leave it to authors to make final decision on this.

2) Fig. 9: Please change the x axis title as "Magnolol concentration" as the current version is a little confusing.

3) Also, I recommend adding a concluding paragraph or two summarizing the overall achievements of the study.

Author Response

Replies to Reviewer 2
We highly appreciate the important comments from the reviewer. We have amended the manuscript according to the comments. Here are the point-to-point replies:

The manuscript submitted by Li et al. is devoted to development and validation of an LC-MS/MS based method of quantitation propylthiouracil and its main metabolite glucuronide. This is the first method to determine both the compounds.
Having read the manuscript, I found that the experiment is very well planned and carried out. All data support the conclusions.
I have a couple of recommendations mentioned below.
1) From my point of view, using molar concentrations such as mM, μM etc. is more inconvenient for analysts who deal routine analyses. I would advise using mass concentrations (ng/mL. mcg/ml etc.) throughout the manuscript. Nevertheless, I leave it to authors to make final decision on this.

We absolutely agree with the reviewer and know the molar concentration is not commonly used in routine analyses. However, in metabolic examination, molar unit is more commonly used to display experimental data. So, we decide to use molar concentration in this manuscript. However, in our next step of investigation, when analyzing the plasma / urine samples from clinical study, we will display our data using mass unit as suggested.

2) Fig. 9: Please change the x axis title as "Magnolol concentration" as the current version is a little confusing.

We have amended the manuscript as suggested.

3) Also, I recommend adding a concluding paragraph or two summarizing the overall achievements of the study.

We have amended the manuscript as suggested (lines 415 – 421):
A HPLC-MS/MS method for the simultaneous quantitation of PTU and its metabolite (PTU-Glu) was successfully developed and validated. This reliable bioanalytical protocol was subsequently applied to examine the in vitro metabolism of PTU. Glucuronidation of PTU was confirmed in pooled human HLMs while UGT1A9 was identified as an important UGT isoform responsible for the glucuronidation of PTU. The information obtained from this study will facilitate future mechanistic investigation on the hepatoxicity of PTU and may optimize its clinical application. Further investigation on this topic is warranted.

Round 2

Reviewer 1 Report

The manuscript in the present form can be published.

Author Response

Thanks