What’s the purpose of this study? Was it to develop a method for the determination of ETS in urine? Several methods have been developed using dilution to eliminate matrix effects in urine. The 150 μL and 300 μL urine sample were used, which were larger than the sample volume used in many literature. LOQ was 0.1 μg/mL, which provided lower sensitivity compared with previous methods. The authors should discuss the novelty and improvement compared to existing methods.

If this study was aimed to compare the matrix effects using orthogonal and z-spray ion sources, same sample preparation and column system should be used. However, this study used two different columns and different dilution methods.

Other issues:
1. Abstract: Dilution method was used for the sample preparation should be mentioned.
2. Page 7: “The accuracy values of the stability study of ethyl glucuronide and IS stock solutions (n= 6) were found to be 1102.5% and 106.3% accuracy.” Please check the number 1102.5%.

1. Chromatographic separation plays an important role in eliminating matrix effects. In the manuscript, the first method was developed by using an Acquity UPLC and Xevo TQD triple quadrupole mass spectrometer, equipped with a UPLC HSS C18 (1.8 μm, 2.1 × 150 mm) column, while the second method was performed on Agilent 1200 series HPLC system coupled to an Agilent 6410B triple quadrupole tandem mass spectrometry with a Zorbax Eclipse Plus C18 column.

In the response letter, the authors stated: “Since the z-spray system includes UPLC when choosing a column, we could not use a column with the same characteristics even though we used a C18 column as a column in both studies.” However, it still does not provide a good explanation as to why the same columns are not used when comparing orthogonal and z-spray ion sources.

1. The authors discussed only literature with limits of detection greater than 0.1 ppm. In fact, there are methods reported which can provide higher sensitivity. The novelty of this article and its improvement on existing methods remains questionable.

Page 20, “In a study in the literature, a 10-fold dilution was made and the LLOQ value of the study was 100 μg/mL14”. Please check the number.