Author's response to reviews

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1 Author's response to reviews Title: Influence of the relative composition of trace elements and vitamins in physicochemical stability of total parenteral nutrition formulations for neonatal use Authors: Bianca W Lobo MSc (biancawaruar@yahoo.com.br) Venício F da Veiga MSc (veigavf@micro.ufrj.br) Lucio M Cabral DSc (lmcabral@pharma.ufrj.br) Ricardo C Michel DSc (rmichel@ima.ufrj.br) Nádia M Volpato DSc (nadia.volpato@ufrgs.br) Valeria P. de Sousa DSc (valeria@pharma.ufrj.br) Version: 2 Date: 15 February 2012 Author's response to reviews: see over

2 Dear Editor: Please find enclosed the revised paper entitled Influence of the relative composition of trace elements and vitamins in physicochemical stability of total parenteral nutrition formulations for neonatal use for your consideration as a regular paper for Nutrition Journal. As you will note, we accepted the reviewer suggestions that are marked in red in the text. We thank the reviewer for their suggestions that significantly improved this article and hope that this revised paper may be accepted by the Nutrition Journal. Sincerely, V. P. Sousa

3 Answers to the referee 1: We appreciate the important suggestions of the reviewer which led to the improvement of the manuscript. Major Compulsory Revision 1. Please check correctness and accuracy of information about dynamic light scattering throughout the manuscript. For example: a. Methods/Dynamic light scattering: What do you mean with Z medium? Assuming that the z-average is mentioned here, note that this size value (together with the polydispersity index) is not a result of CONTIN analysis. We used the equipment Malvern Zetasizer 3000HS in monomodal (or cumulant analysis) mode. As pointed by the referee the values were expressed with wrong nomenclature. It is z- average not z-medium as previous stated. Attached (at the end) is an example of analytical report obtained in the DLS analysis. The manuscript was revised (lines ). b. Results/Globule size determination /2nd paragraph: Polydispersity index was less than 0.7 Such a high value indicates distinct inhomogeneities in thesamples. Please specify the polydispersity indices for the different measurements (e.g. in table 3). The reasons for unusual high values (above 0.3) should be discussed. The polydispersity values were reviewed and it was found that were less than 0.3, as now showed in Table 3. Thus, the dispersions were found to be quite homogeneous. The phrase "Polydispersity index was less than 0.7" was changed to "the polidispersity index was less than 0.3 for all samples, as presented in Table 3, showing homogeneity of the dispersions" (lines ). c. Results/Globule size determination /2nd paragraph: Please correct: DLS,are capable of detecting globule diameters of ( µm). The upper size limit for DLS measurements is about 1-5 µm. Check the cited references. Please check also the 1st sentence of the 4th paragraph ("The great limitation...") for accuracy. As mentioned by the referee, the instrument used (Zetasizer 3000HS) is able to detect particles from 1 nm to 3 µm. References were checked. Based on the information given in

4 the manual of the equipment the phrase was changed to The instrument used for particle size distribution analysis is capable of detecting a wide range of lipid globule diameters of 1 nm to 3 µm. (lines ). d. Results/Globule size determination /last paragraph: and Laser Diffraction Methods, as DLS, Laser diffraction (static light scattering) should not be mixed up with dynamic light scattering. Although based on light scattering, the measurement principles (and thus measurement size range, advantages and limitations, size information) are different. For information about principle of static light scattering see for example H.G. Merkus, Particle Size Measurements:Fundamentals, Practice, Quality (Springer/2009); C. Washington: Particle size analytics in pharmaceutics and other industries (Ellis Hoorwood/1992); J. Kuntsche et al., J.Biomed.Nanotechnol. 5 (2009) 384. The nomenclature of the method applied in this work was described incorrectly, thus the term "Laser Diffraction" was replaced by "Light Scattering Methods" (Dynamic Light Scattering - DLS) (line 434). The suggested references were considered. 2. Interpretation of the size results obtained by optical microscopy should be done with more care. Light microscopy is a valuable tool for the detection of larger droplets in µm-size range which are not detectable, for example, by dynamic light scattering. However, its usefulness for establishing size distributions or a mean droplet size in colloidal formulations appears a bit questionable. Results shown in table 3 clearly show limitations of light microscopy for this approach: The average diameters vary considerably (i.e. from 400 to 870 nm for formulation NP1) and also the SD values indicate large variations between the single determinations of the same sample. Please add information about repeatability and accuracy of this method for size determinations of colloidal fat emulsions. Information about the largest detected droplet size in the formulations after different storage time/temperature would be desirable. As correctly suggested by the referee, the discussion was expanded in Results and Discussion, section Globule size determination by DLS, OM and LO (lines ). The largest droplet sizes detected in the formulations are presented in the Table Results/Visual inspection.../2nd paragraph: "... cream layer is visible as a translucent band separated from the remaining TPN..." Check for correctness! A translucent layer indicates presence of oil on the top on the emulsion (phase separation/cracking, see i.e. reference 26). This is an irreversible process and one

5 should expect an alteration in the fraction of larger emulsion droplets (# 5 µm) in this case. Detailed information about the sampling procedure needs to be added to the manuscript. How it was assured that a homogeneous sample (representative for the whole) has been withdrawn from the infusion bags? During the visual inspection it was observed alteration in the formulations similar to that described at Gonyon et al., 2007 (reference 26). The alteration observed is described as creaming, as an opaque white layer in the sample surface (lines ). The samples homogeneity was assured by the agitation before the sampling procedure (lines ). Minor Essential Revisions 4. Introduction/4th paragraph: Please check for accuracy/correctness: individual lipid particles must be between 0.4 µm to 1 µm in size. The mean droplet diameter of colloidal fat emulsions is usually even smaller than 400 nm (see manuscript/results/globule size determination.../3rd paragraph and cited references). Furthermore, "... where GSD (globule size distribution) cannot exceed 500 nm..." should be "... where the mean droplet size should not exceed 500 nm...". As correctly suggested by the referee the text was changed. The lipid droplet size must be within 0.25 and 0.5 µm to ensure homogeneity and being consistent with the chylomicrons size (lines 42-45). 5. There are discrepancies in the composition of the formulations between the manuscript (Material and Methods, Composition of the admixtures) and Table 1: zinc acetate/sulfate, sodium chloride/hydrochloride, magnesium sulfide/sulfate. Please check and clarify! The discrepancies in the composition of the formulations between the manuscript and Table 1 were corrected (Table 1 and lines 77-79). 6. Methods/DLS: Check correctness of cited reference "... according to the equipment manual (26)..."Improve expression of the sentence "... the measurements taken were specified by international standards..."? What stands "[MP1]" for? We corrected the reference. The manuscript was reviewed (lines ). "[MP1]" was an alteration made by the English reviewer and was removed.

6 7. In particle size determinations of colloidal formulations, potential contamination with dust particles may cause problems. This is particularly important in single particle counting. Have required precautions (e.g. filtration of all diluents) been taken for the size determinations in this study? Please add this information in the method section. The procedure was now better described (lines ). 8. Results/Zeta potential: The second paragraph is a bit confusing to read and should be improved. Furthermore, the influence of the zeta potential on the stability of parenteral fat emulsions as well as destabilization by additives has already been studied intensively. The authors may consider the literature data, for example: C. Washington, Int.J.Pharm. 66 (1990) 1-21; C. Washington et al., Int.J.Pharm. 54 (1989) ; C. Washington, Int.J.Pharm. 87 (1992) ). Please check for accuracy (2nd paragraph): this parameter (zeta potential) reveals the distance between the emulsifier s charge and the lipid droplets... As correctly mentioned by the referee, the paragraph was changed and the discussion improved (lines ). 9. There were significant differences in the PFAT between formulations NP1/NP3 and NP2 (table 3). The reasons for this result should be discussed in the manuscript. This result may be related to the absence of vitamins in this formulation, as only in the formulations NP1 and NP3 the vitamin is present. This discussion was added in the text (lines ). 10. Results/Globule size determination.../end of 7th paragraph: What do you mean with "... electronic microscopy..."? The correct term is electron microscopy (line 383). 11. Results/Globule size determination.../last paragraph: "... while European Pharmacopoeia indicates only OM for sub-visible particulate matter..." The

7 actual edition of the European Pharmacopoeia includes light obscuration particle counting for the evaluation of sub-visible particulate matter in injections and infusions even as the preferred method over optical microscopy (see Ph.Eur Particulate contamination: Sub-visible particles; Method 1). Please correct! As correctly mentioned by the referee, the European Pharmacopoeia recommends light obscuration and light microscopy to evaluate particulate contamination in sub-visible particles. We corrected it (lines ). 12. Figure 1: Eliminate the asterisks indicating significant differences in this figure as the differences are likely not significant (as stated in the manuscript, results/zeta potential determination/1st paragraph). We corrected it. 13. Table 3: Please specify what size value is given in the table for optical microscopy; median (D50) obtained from a size distribution as stated in the manuscript (Globule size determination.../7th paragraph)? Preferably, the z-averages and polydispersity indices obtained by DLS should be added to the table. It was added in Table 3 footnote indicating the size value by OM. The polidispersity indices were added to Table 3. Discretinoary Revisions 14. Organization of table 1 is a bit confusing and should be improved. Preferably the added volumes of stock formulations should be specified together with the concentration of the active ingredients in the final formulation. All concentrations should refer to the same unit (e.g. amount active ingredient [g or Eq] per mass or volume of final formulation) and should not be given as a dose. Does the value given for the vitamins refer to the added volume of stock solution (MVI 12 opoplex)? The table 1 was improved. Instead of dose were expressed concentration of nutrients in the formulation and volumes of stock solutions used. The vitamin complex used was Opoplex MVI Results/pH evaluation/1st paragraph:... NP3 admixture, which displayed incompatibility due to. Was this incompatibility detected during the study?

8 Otherwise which displayed should be replaced by which may be prone to or similar. It was not detected incompatibility. It was added the sentence: Even the NP3 admixture, which could be prone to incompatibility, due to a possible interaction between vitamins and trace elements, did not have any variation in ph value. (lines ). 16. Results/Globule size determination.../end of 12th paragraph: What do you mean with "... storage conditions... recommended for technical procedures and political policy,..."? The correct terms are Brazilian and international guidelines, the references were also included (lines ). 17. Tables 1 and 3 contain essential information and should be added to the manuscript and not given as supplementary information. These tables, despite being presented in a separate file, are part of the manuscript.

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