Accurate and Sensitive UHPLC–Tandem Mass Spectrometry Sequential Methods for Therapeutic Drug Monitoring of Aztreonam/Avibactam in Human Plasma

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The use of two different columns for a method claimed to analyze 2 drugs may represent a significant analytical limitation. Please provide your comments.

Reference #27: The most recent document for the validation of bioanalytical methods is ICH M10 on Bioanalytical Method Validation – Scientific Guideline, available at https://www.ema.europa.eu/en/ich-m10-bioanalytical-method-validation-scientific-guideline.

 

The inclusion of data related to incurred sample reanalysis (as required by the latest ICH guidelines) could add value to the present study."

Author Response

Dear Editor,

We would like to thank you for the opportunity to resubmit a revised version of this manuscript. We appreciated the reviewer’s constructive comments. All have been carefully considered and incorporated, where and whenever possible, in the revision.

Our point-by-point responses are provided below.

Q= QUERY; A= ANSWER

Reviewer #1:

Q1. The use of two different columns for a method claimed to analyze 2 drugs may represent a significant analytical limitation. Please provide your comments.

A1. We thank the reviewer for this comment, allowing us to better clarify this issue. The use of two different stationary phases was a deliberate analytical choice justified by the markedly different physicochemical properties of aztreonam and avibactam. Despite both being hydrophilic β-lactam–related compounds, they exhibit distinct polarity, ionization behavior, and retention characteristics. The use of a Poroshell EC-C18 column for aztreonam and a Poroshell PFP column for avibactam allowed optimal retention, improved peak symmetry, enhanced selectivity, and reproducible retention times under fast gradient conditions. Importantly, both analytes were processed using the same sample preparation protocol and analyzed within the same analytical platform (UHPLC–qTOF MS/MS), ensuring workflow consistency. In routine TDM practice, the use of different chromatographic methods within the same platform does not represent a significant operational limitation, especially when balanced against improved analytical robustness and sensitivity. We discussed this issue in the Discussion section of the manuscript (refer to Line 399-411).

Q2. Reference #27: The most recent document for the validation of bioanalytical methods is ICH M10 on Bioanalytical Method Validation – Scientific Guideline, available at https://www.ema.europa.eu/en/ich-m10-bioanalytical-method-validation-scientific-guideline.

A2 We thank the Reviewer for highlighting this important regulatory update. The manuscript has been revised according to the updated version of the ICH M10 Guideline on Bioanalytical Method Validation. The reference list has been modified accordingly (Ref. 27).

Q3. The inclusion of data related to incurred sample reanalysis (as required by the latest ICH guidelines) could add value to the present study.

A3. We thank the Reviewer for this valuable comment. We fully acknowledge that ISR represents an important component of bioanalytical method validation in the context of regulatory clinical trials and pivotal pharmacokinetic studies. However, the primary aim of the present work was the development and analytical validation of a UHPLC–qTOF MS/MS method intended to support therapeutic drug monitoring in routine clinical practice, rather than to generate pharmacokinetic data within a regulated clinical trial framework. The clinical samples included in this study were used exclusively to demonstrate real-world applicability of the validated analytical method. The number of available samples was limited and derived from clinical activity, which did not allow structured ISR implementation. Moreover, full validation was performed in accordance with international bioanalytical guidelines, including assessment of accuracy, precision, selectivity, matrix effects, recovery, stability, and carry-over, which collectively demonstrate the robustness and reproducibility of the method. Considering the methodological aim of the manuscript and the non-pivotal nature of the clinical application presented, we believe that ISR, while valuable in confirmatory clinical studies, is not strictly required in this specific context of analytical method validation for TDM support. However, we added this issue among limitations of our study (refer to Line 411-424).

Reviewer 2 Report

Comments and Suggestions for Authors

 

The manuscript has some constraints and vulnerabilities;

1. Why a simultaneous method was not developed for the two analytes.
2. A significant shortcoming of the article is instability of Aztreonam under specific conditions. It is unstable under third freeze-thawed cycle and on borderline after 2^nd While 3^rd is not within range. Some measures or alternative method for stability needs to be adopted. Are such stability problem have been reported? If not then it is a huge limitation of the study.
3. Table 10, why day 05 stability was not evaluated. It seems analyte is less stable. The stability of sample extracts is limited. Aztreonam extracts are stable for only up to three days when kept at 4°C, whereas avibactam is stable for up to five days. This limited window could restrict the ability to re-analyze samples later.
4. Both analytes exhibited significant matrix effects. Aztreonam showed somewhat signal suppression., while avibactam showed a signal increase. The IS may effectively counter these effects but still the patient samples will be prone to matrix effects.

 

Other minor concerns are as

1. Line 158 and 159, use symbol instead of “Micron”
2. Table 3 and 4, please clarify what “CAD” for?
3. Line 184, LLoQ, no need to write “o” in lower case.
4. Line196- 197, “if the correlation coefficient (R²) was ≥ 0.998” please provide reference for this statement. ≥ 0.999 is generally accepted, but for many HPLC assays, ≥ 0.999 is expected.
5. Section 2.5.2, line 198 “2 µg/mL” LOQ of one analyte is provided, please provide the LOQ of the other analyte as well.
6. Section 3.1, optimization of mobile phase composition and stationary should be provided.
7. Line 373, “Specifically, thanks to the high accuracy in…..”, please use standard writing format.
8. Line 376, “detection limits are significantly lower for both analytes”, detection limit or limit of detection (LOD), is a different approach. The authors in their result have focused on LOQ. It may be replaced with “quantification limit”
9. Please provide information about the variation in concentration of patients. What are the optimum levels/concentration required for effective antimicrobial activity. How much of the patients did not achieved the therapeutic levels?
10. The number patients are not enough.
11. Also provide the timing of sample collection from the patients

Comments on the Quality of English Language

The English is fine but in some places authors need to adopt standard manuscript writing style.

Author Response

Dear Editor,

We would like to thank you for the opportunity to resubmit a revised version of this manuscript. We appreciated the reviewer’s constructive comments. All have been carefully considered and incorporated, where and whenever possible, in the revision.

Our point-by-point responses are provided below.

Q= QUERY; A= ANSWER

Reviewer #2:

The manuscript has some constraints and vulnerabilities.

We sincerely thank the Reviewer for the thorough evaluation of our manuscript and for highlighting important methodological aspects. Each concern has been carefully considered and is addressed below.

 

Major concerns

Q1. Why a simultaneous method was not developed for the two analytes.

A1. We thank the reviewer for this comment, allowing us to better clarify this issue. According to the markedly different physicochemical and chromatographic behaviors of aztreonam and avibactam, optimal retention and peak shape for both analytes could not be achieved under identical chromatographic conditions. Aztreonam exhibited adequate retention and peak symmetry on a C18 stationary phase, whereas avibactam showed insufficient retention and peak distortion under the same conditions. Conversely, chromatographic conditions optimized for avibactam resulted in suboptimal performance for aztreonam. Furthermore, our qTOF platform is unable to simultaneously change polarity, and the two analytes are analysed respectively in positive (aztreonam) and negative (avibactam) electrospray ionization, as this provides greater sensitivity and signal stability under our chromatographic conditions. For this reason, two sequential methods were implemented on the same UHPLC–qTOF MS/MS platform, using identical sample preparation and analytical workflow. We discussed this issue in the Discussion section of the manuscript (refer to Line 399-411).

Q2. A significant shortcoming of the article is instability of Aztreonam under specific conditions. It is unstable under third freeze-thawed cycle and on borderline after 2nd While 3rd is not within range. Some measures or alternative method for stability needs to be adopted. Are such stability problem have been reported? If not then it is a huge limitation of the study.

A2. We thank the Reviewer for raising this critical point. The observed instability of aztreonam after repeated freeze–thaw cycles is consistent with the known chemical reactivity of β-lactam antibiotics. Aztreonam contains a β-lactam ring that is susceptible to hydrolytic degradation, particularly under repeated thermal stress conditions. Similar stability limitations have been reported for other β-lactam antibiotics in bioanalytical studies and therapeutic drug monitoring settings. Importantly, aztreonam remained stable after one freeze–thaw cycle, a borderline deviation was observed after the second cycle and degradation outside ±15% occurred after the third cycle. This allowed to reanalyze sample up to two freeze-thaw cycles. However, this does not represent an unexpected analytical artifact, but it reflects intrinsic chemical instability of the molecule. Importantly, ICH M10 requires stability assessment but does not mandate stability beyond clinically realistic handling conditions. Indeed, during routine TDM-guided practice, the samples are typically processed immediately or stored/frozen and subjected to a maximum of one freeze-thaw cycle. To mitigate degradation risk, we recommend analyzing the sample immediately after collection from the patient. If this is not possible, it is preferable to centrifuge it to isolate the plasma and store it at −80 °C, so that it can be analyzed after only one freeze-thaw cycle. Data regarding the stability of aztreonam were presented in section ‘3.2. Method Validation’ (Line 325-331), while we discussed the issue of stability in section ‘4. Discussions’ (Line 398-400).

Q3. Table 10, why day 05 stability was not evaluated. It seems analyte is less stable. The stability of sample extracts is limited. Aztreonam extracts are stable for only up to three days when kept at 4°C, whereas avibactam is stable for up to five days. This limited window could restrict the ability to re-analyze samples later.

A3. We appreciate this comment. Day 5 stability was evaluated for avibactam extracts because preliminary stability screening suggested longer stability under refrigerated conditions. In contrast, early pilot experiments indicated that aztreonam showed significant degradation beyond day 3 at 4°C; therefore, extended evaluation to day 5 was not pursued, as concentrations were already outside acceptable ranges. The limited extract stability of aztreonam (3 days at 4°C) is acknowledged in the manuscript and reflects its intrinsic chemical lability. However, in routine laboratory workflow, clinical samples are analyzed within 24–48 hours and extract reanalysis rarely exceeds 72 hours. If necessary, long-term storage is performed at −80°C prior to extraction. Therefore, the 3-day stability window is compatible with routine TDM-guided practice.

Q4. Both analytes exhibited significant matrix effects. Aztreonam showed somewhat signal suppression., while avibactam showed a signal increase. The IS may effectively counter these effects but still the patient samples will be prone to matrix effects.

A4. We thank the Reviewer for this technically relevant observation. Matrix effects are commonly observed in LC–MS/MS bioanalysis, especially for highly polar compounds analyzed by electrospray ionization. However, the key parameter under ICH M10 is the IS-normalized matrix effect, which was within ±15% at all QC levels for both analytes. The use of stable isotopically labelled internal standards ([²H₆]-aztreonam and [¹³C₅]-avibactam) effectively compensated for ionization variability. By treating patient samples in the same way as calibrators (precipitation in methanolic solution containing internal standards), any matrix effect present in them is fully controlled and corrected thanks to the presence of IS. Indeed, clinical samples quantified fell within validated ranges.

Minor Concerns

Q1. Line 158 and 159, use symbol instead of “Micron”

A1. We thank the Reviewer for this correction. The term “Micron” has been replaced with the symbol “µm” in Line 161 and 162.

Q2. Table 3 and 4, please clarify what “CAD” for?

A2. Thank you for this suggestion. “CAD” refers to Collisionally Activated Dissociation gas. This has now been explicitly clarified in the table footnotes to avoid ambiguity.

Q3. Line 184, LLoQ, no need to write “o” in lower case.

A3. Thank you for this comment. The acronym has been standardized to LLOQ throughout the manuscript.

Q4. Line196- 197, “if the correlation coefficient (R²) was ≥ 0.998” please provide reference for this statement. ≥ 0.999 is generally accepted, but for many HPLC assays, ≥ 0.999 is expected.

A4. We thank the Reviewer for this important observation. The statement has been revised (Line 199-204) to align with ICH M10 guidelines, which emphasize accuracy and precision of calibration standards rather than a fixed R² threshold. The text has been modified to clarify that calibration curve acceptance was based on back-calculated concentrations meeting ±15% (±20% at LLOQ), in accordance with ICH M10. The R² value is now reported descriptively rather than as a strict acceptance criterion.

Q5. Section 2.5.2, line 198 “2 µg/mL” LOQ of one analyte is provided, please provide the LOQ of the other analyte as well.

A5. We thank the Reviewer for this observation. We stated the LLOQ values for both analytes in Section 2.5.2 (Line 205).

Q6. Section 3.1, optimization of mobile phase composition and stationary should be provided.

A6. We thank the Reviewer for this comment. Method development included preliminary optimization of chromatographic conditions using the stationary phases and mobile phase compositions available in our laboratory. The selection of the final chromatographic conditions was guided by practical analytical criteria, including retention time, peak symmetry, signal intensity, and overall reproducibility. Given the distinct physicochemical properties of aztreonam and avibactam, the final conditions were chosen to ensure adequate retention and optimal peak shape for each analyte, while maintaining a short analysis time and compatibility with high-resolution MS detection. The aim of the study was to validate a robust and applicable bioanalytical method rather than to perform a systematic comparison of multiple stationary phases or mobile phase systems. For clarity, a brief statement has now been added in Section 3.1 (Line 257-264) to explain the rationale for the selected chromatographic conditions.

Q7. Line 373, “Specifically, thanks to the high accuracy in…..”, please use standard writing format.

A7. The sentence has been revised to conform to standard scientific writing style and improve clarity (Line 383-385).

Q8. Line 376, “detection limits are significantly lower for both analytes”, detection limit or limit of detection (LOD), is a different approach. The authors in their result have focused on LOQ. It may be replaced with “quantification limit”.

A8. We thank the Reviewer for this precise observation. The terminology has been corrected. The expression “detection limits” has been replaced with “quantification limits” (Line 386) to ensure consistency with the validated parameter.

Q9. Please provide information about the variation in concentration of patients. What are the optimum levels/concentration required for effective antimicrobial activity. How much of the patients did not achieved the therapeutic levels?

A9. We thank the reviewer for this comment, allowing us to better clarify this issue in the context of the aim of our manuscript. The variations observed in concentrations of both aztreonam and avibactam could be justified by the variation in renal function among patients, considering the hydrophilic features of both agents and their predominant renal clearance. The optimal target for this agent was a 60%fT_>MIC for aztreonam coupled with 50%fT > 2.5 mg/L for avibactam according to preclinical models (refer to 10.1128/aac.01950-24). However, this information are beyond the scope of this paper. Similarly, the prevalence of patients which failed in attained the optimal PK/PD target was not assessed considering that it was out of the scope of the paper. Indeed, our manuscript aimed to develop and validate two rapid, accurate, and sensitive UHPLC–qTOF MS/MS sequential methods for quantifying aztreonam and avibactam in human plasma, suitable for routine clinical TDM.

Q10. The number patients are not enough.

A10. We acknowledge that the patient number (n=20) is limited. However, these samples were not intended to support clinical conclusions but only to demonstrate method applicability to plasma samples collected during routine clinical practice. For analytical validation purposes, method performance characteristics (precision, accuracy, matrix effect, recovery, stability) are independent of patient sample size.

Q11. Also provide the timing of sample collection from the patients

A11. We thank the reviewer for this comment, allowing us to better clarify this issue in the context of the aim of our manuscript. Indeed, our manuscript aimed to develop and validate two rapid, accurate, and sensitive UHPLC–qTOF MS/MS sequential methods for quantifying aztreonam and avibactam in human plasma. According to this objective, the timing of sample collection from the patients is not a mandatory information for validating the analytical method.

Reviewer 3 Report

Comments and Suggestions for Authors

A combination of aztreonam and avibactam is a new promising antibacterial medication and its brand name is Emblaveo. The manuscript pharmaceutics-4191179 is well written and contains elements of scientific innovation in the field of LC-MS analysis of aztreonam and avibactam. I suggest its slight modification in line with the following comments.

1. Figure 1 – the structures of aztreonam and its isotope labelled conjugate should be drawn in an uniform manner (e.g. thiazole ring, stereochemistry).
2. Figure 2 – the structure of isotope labelled avibactam should be drawn more clearly.
3. Line 242 – “…MS/MS fragmentation pattern spectra of the analytes and by comparing them with those reported in the literature [21,23,28]. The specific MRM transition parameters are summarized in Table 5.” However, none of these references contain MS/MS data of avibactam in the negative ion mode as it is in the pharmaceutics-4191179 (in [23] the avibactam has been analyzed in the positive ion mode). Can the authors support the formation of the claimed product ion at m/z 96 (Table 5)?
4. Citation of references [21] and [28] should be completed (e.g. lack of the article number).
5. What about the limit of detection?

Author Response

Dear Editor,

We would like to thank you for the opportunity to resubmit a revised version of this manuscript. We appreciated the reviewer’s constructive comments. All have been carefully considered and incorporated, where and whenever possible, in the revision.

Our point-by-point responses are provided below.

Q= QUERY; A= ANSWER

Reviewer #3:

A combination of aztreonam and avibactam is a new promising antibacterial medication and its brand name is Emblaveo. The manuscript pharmaceutics-4191179 is well written and contains elements of scientific innovation in the field of LC-MS analysis of aztreonam and avibactam. I suggest its slight modification in line with the following comments.

We thank the reviewer for appreciating our manuscript. Each point has been addressed as detailed below.

 

Q1. Figure 1 – the structures of aztreonam and its isotope labelled conjugate should be drawn in an uniform manner (e.g. thiazole ring, stereochemistry).

A1. We thank the Reviewer for this important observation. The chemical structures in Figure 1 have been revised to ensure graphical uniformity between aztreonam and its isotope-labelled analogue.

 

Q2. Figure 2 – the structure of isotope labelled avibactam should be drawn more clearly.

A2. We appreciate the Reviewer’s comment. Figure 2 has been revised to improve clarity of the isotope-labelled avibactam structure.

 

Q3. Line 242 – “…MS/MS fragmentation pattern spectra of the analytes and by comparing them with those reported in the literature [21,23,28]. The specific MRM transition parameters are summarized in Table 5.” However, none of these references contain MS/MS data of avibactam in the negative ion mode as it is in the pharmaceutics-4191179 (in [23] the avibactam has been analyzed in the positive ion mode). Can the authors support the formation of the claimed product ion at m/z 96 (Table 5)?

A3. We thank the Reviewer for this insightful comment. Although it is correct that some of the cited references report avibactam analysis predominantly in positive ion mode, in our method avibactam was analyzed in negative electrospray ionization, which provided superior sensitivity and signal stability under our chromatographic conditions. The precursor ion observed in negative mode corresponds to the deprotonated molecular ion [M–H]⁻ at m/z 264.0308. Collision-induced dissociation experiments were systematically performed during method development, and the product ion at m/z 95.9528 (reported in Table 5) was found to be the most abundant and reproducible fragment under optimized collision energy conditions. This fragment is consistent with cleavage of the diazabicyclooctane core and formation of a stable sulfonate-derived anion fragment. The accurate mass measurement obtained with the qTOF platform supports the elemental composition of this fragment ion within mass accuracy < 5 ppm, confirming its structural plausibility.

 

Q4. Citation of references [21] and [28] should be completed (e.g. lack of the article number).

A4. We thank the Reviewer for identifying this oversight. References [21] and [28] have been carefully revised according to journal formatting requirements.

 

Q5. What about the limit of detection?

A5. We appreciate this comment. In accordance with current bioanalytical method validation guidelines, the lower limit of quantification (LLOQ), rather than the limit of detection (LOD), is considered the relevant parameter for quantitative bioanalytical methods intended for clinical application. Given that the method was specifically developed for accurate quantification within clinically relevant concentration ranges, emphasis was placed on establishing and validating the LLOQ (0.2 µg/mL for aztreonam and 0.1 µg/mL for avibactam), which met accuracy and precision acceptance criteria. Nevertheless, for completeness, the estimated LOD values calculated based on signal-to-noise ratio (S/N ≈ 3) and extrapolated from the experimental S/N obtained at LLOQ level, were approximately 0.008 µg/mL for aztreonam and 0.014 µg/mL for avibactam.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have rectfied the manuscript. It is improved and easy to understand for the readers. 

Comments on the Quality of English Language

The English is fine but in some places authors need to adopt standard manuscript writing style.