Abdelmajed et al. BMC Chemistry(2024) 18:158
https://doi.org/10.1186/s13065-024-01261-9

BMC Chemistry

RESEARCH

Open Access

Full green assay of parenteral dosage formsof polymyxins utilizing xanthene dye:application to content uniformity testing
Mahmoud A. Abdelmajed1*, Khalid M. Badr El-Din2, Tamer Z. Attia2* and Mahmoud A. Omar2,3
Abstract
Due to the lack of other treatment options, a rebirth of polymyxins is urgently required. Colistin (also calledpolymyxin E) and polymyxin B are the only two examples of this antibiotic class that were effectively employedin such critical situations. In the present work, both of the two studied medications were quantified via a simple,
green, and non-extracting spectrophotometric approach based on the formation of ion-pair complexes with
Erythrosine B. Without using any organic solvents, the pink color of the created complexes was detected atwavelength = 558 nm. To achieve the highest intensity of absorbance, optimum conditions were establishedby the screening of many experimental factors such as pH, buffer volume, the volume of Erythrosine B, andthe time consumed to undergo the reaction. For Colistin and Polymyxin B respectively, Beer-Lambert’s law wasobserved at the concentration ranges of 1–6, 1–9 µg mL− 1. The technique was approved and validated following
ICH recommendations. Lastly, the suggested approach has been successfully implemented to quantify the cited
medications colorimetrically, for the first time, in their parenteral dosage forms with excellent recoveries. Also,
Content uniformity testing was implemented.
Keywords Polymyxins, Colistin (polymyxin E), Polymyxin B, Erythrosine B, Parenteral dosage forms, Content
uniformity testing*Correspondence:
Mahmoud A. Abdelmajed
Mahmoud.alaa81@yahoo.com
Tamer Z. Attia
tamer_zekry_a@yahoo.com
1
Department of Pharmaceutical Analytical Chemistry, Faculty of
Pharmacy, Deraya University, New Minia, Egypt
2
Department of Analytical Chemistry, Faculty of Pharmacy, Minia
University, Minia, Egypt
3
Department of Pharmacognosy and Pharmaceutical Chemistry, Collegeof Pharmacy, Taibah University, Medinah, Saudi Arabia

Introduction
With the emergence of multidrug-resistant (MDR)
Gram-negative bacteria and the lack of innovative antimicrobial medications, the revival of polymyxins (PMS)
is urgently essential [1, 2]. Recently, there has been a significant rise in infections caused by MDR Gram-negative
pathogens, named Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae. For thesesubtypes of bacteria, PMS are frequently the only active
antibiotics and are remarkably successful in killing them.
Colistin (CS) and Polymyxin (Poly B) have relatively similar chemical structures. The only difference between thetwo structures occurs in one amino acid at position 6 ofthe peptide ring, where CS has a d-leucine and Poly B has
a d-phenylalanine, as observed in (Fig. 1). Moreover, the© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 Chemical structure of Colistin (a) and Polymyxin B (b)way that both drugs are delivered into the human body isalso one of their main differences. While Poly B is given
directly as its active form, CS is taken as an inactive lesstoxic prodrug called Colistimethate Sodium (CMS) [3].
Numerous analytical strategies based on spectroscopic (spectrophotometric [4–8] and fluorimetric[9–14] methods and also microbiological assays [15–19]
were reported. On the other hand, chromatographic
approaches include high-performance liquid chromatography (HPLC) [20–26], liquid chromatography coupled with mass detector (LC-M) [27–36], and capillaryelectrophoresis (CE) [37–40], also published. As wellknown, separative techniques such as HPLC and LC-M
consume large amounts of energy, need well-experienced
operators, and have complex and costly instrumentation.
Microbiological assays need a long incubation time andlack selectivity and sensitivity. Belongs to the reported
spectrophotometric approaches [5–8] were less sensitive
than the present work. Also, the complex formation with
FeCl3 [4] needed an organic solvent (methanol) but thepresent study has been completely performed in aqueous
media. Related to the fluorimetry technique, three articles were laborious and time-consuming [9–11]. And theother needed a costly reagent and organic solvent (acetone) to proceed [12]. Consequently, the valuable aim of
the current work was to establish a rapid, sensitive, costeffective, and green alternative technique for analyzingthese drugs. Moreover, it is the first colorimetric paper

employed to quantify the parenteral dosage forms of theinvestigated drugs, along with the employment of Content uniformity testing and greenness evaluation.
Ion pair complex formation is one of the most applied
spectroscopic approaches that depend on the coloredproduct measurement in the visible region. Therefore,
the final colored product is devoid of any interfering
substances such as any solvent or degradation product.
Erythrosine B (EB) is a xanthene dye that is widely used
as a spectroscopic probe in the investigation of pharmaceutical and biological substances [41]. It is one of themost commonly employed reagents in ion-pair complex
reactions in a simple methodology. Chemically, it is adisodium salt of 2, 4, 5, 7-tetraiodofluorescein having anegative charge that contributes to the formation of thecolored complex together with the positive charge of theexperimental substances.

Experimental
Devices

All colorimetric measurements were performed using aspectrophotometer made by Shimadzu UV-visible 1900i(Tokyo, Japan). Two quartz sample cells (1 cm) were
utilized to measure the absorbance. A digital analytical balance (Mettler Toledo, Glattbrugg, Switzerland), a
thermostatically controlled MLW type water bath (Memmert Gmbh, Schwabach, Germany), and an ADWA 11

Abdelmajed et al. BMC Chemistry(2024) 18:158

pH meter (Romania) instruments also were involved in
the described methodology.
Pure powder and marketed formulations

CS and Poly B powders with purity grades of 99.2% and98%, respectively were kindly gifted by the National
Organization for Drug Control And Research (NODCAR) and utilized without further purification.
Paximid® vial (a Cipla Company product) is labeled tocontain 500,000 IU, which is equal to 5 mg of the authentic drug). Colomycin® lyophilized powder for injection(Forest Laboratories, United Kingdom) is labeled to contain 2 M.I.U which is equivalent to 160 mg of CMS, theprodrug of CS.
Chemicals and reagents

EB was obtained from (Loba Chemie, Mumbai, India)
and prepared as 0.15% (w/v) in distilled water (DW).
Drug stock solution

CS and Poly B were daily prepared as a working solutionof 100 µg mL− 1 by weighing 10 mg of each authentic in
100 mL of DW.
Standard analytical procedure

In addition to one mL of the screened medications’working solutions, one mL of acetate buffer (pH = 4) was
added to a 10-mL volumetric flask. Then 0.5 mL of 0.15%
w/v EB was poured. The flasks’ components were kept atroom temperature for ten minutes. Eventually, following
the dilution with DW, the absorbance of each medication
was determined at a wavelength of 558 nm. A blank solution was made in the same way without the addition of
the cited drugs.

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Quantification of CS and poly B in their Market products
Preparation of vial form of the studied drugs

A precise amount from Colomycin® vial equal to 10.0 mg
of the studied medication was poured into a volumetric flask of 100 mL, then five mL of 0.2 M sulfuric acidwas added and remained for ten minutes to convert all
CMS into CS [42]. Furthermore, ten mL of 0.2 M NaOH
was added to neutralize the solution. Belongs to Poly B,
in a 100-mL standard flask, an exact equivalent to 10 mg
of Poly B was moved and completed to mark with DW.
Then, further dilution was employed to obtain working
solutions and previously general analytical procedures
were implemented for both drugs.
Preparation of Colistin Sulfate® tablets

Twenty tablets were weighed, ground, and well-mixed.
After that, an aliquot amount equal to 10.0 mg of CS was
mixed with 30 mL of D.W. and sonicated for 15 min. Filtration and further dilution were employed to provide
the final working solutions. Finally, the general analyticalprocedure was followed.
Procedure for Content Uniformity Testing

During the investigation, 10 tablets of Colistin sulfate®
were individually well-shriveled, from each crushed pill,a specified quantity equal to 10 mg of authentic was
employed and then followed the same steps in Sect. 2.6.2.
Testing for content homogeneity was carried out following USP requirements [43]. The acceptance value (AV)was determined after 10 tablets had each been evaluatedindependently.

Results and discussion
The formed products were water-soluble and no organic
solvents were involved throughout the described
approach. Moreover, the resultant ion-pair complexes
were measured precisely and directly without the necessity for organic solvent extraction. All these features conflict with the environmental friendliness, simplicity, andnot time-consuming of the present work over the otherreported methods. Several medications were spectrophotometrically and spectrofluorimetrically investigatedthrough complex formation with EB [44–50]. In an acidic
media, the reaction between CS and Poly B produced astable, robust, and water-soluble pink complex with amaximum absorbance at λ = 558 nm. The schematic reaction pathway and approach’s spectrum were shown in(Fig. 2) and (Fig. 3) respectively.
Optimization

To find the best reaction circumstances required to produce the highest absorbance readings, certain techniquefactors were varied while the others remained frozen.
Fig. 2 Schematic pathway between polymyxins and Erythrosine B

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Fig. 3 Absorption spectra of ion-pair complexes of Colistin (6 µg mL− 1) and polymyxin B (9 µg mL− 1) with Erythrosine B

Fig. 4 Effect of the pH on the absorbance of the ion-pair complexes of Colistin (3 µg mL− 1) and Polymyxin B (5 µg mL− 1)

Buffer solution

Because any alteration in pH has a significant effect on
the yielded absorbance of the colored complex, a buffer ofacetate was utilized to obtain a pH range (3–5). The bestvalues were in the pH range (3.8–4.2) and any deviation
had an extremely bad effect on the outcomes. During thescreening of the optimal volume of the used buffer, themaximum ABS has been recorded between the quantities(0.5–1.5 mL). As a result, 1 mL acetate buffer (pH 4) was
the best buffer condition, as shown in (Figs. 4 and 5).
Erythrosine B volume optimization

The cited drugs were subjected to varying volumes of thedye ranging from (0.1-1 mL). Turbidity in the experimental solutions was caused by higher dye volumes. Consequently, 0.5 mL of 0.15% w/v EB for both drugs yieldedAbdelmajed et al. BMC Chemistry(2024) 18:158

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Fig. 5 Effect of the buffer volume on the absorbance of the ion-pair complexes of Colistin (3 µg mL− 1) and Polymyxin B (5 µg mL− 1)

Fig. 6 Effect of erythrosine B volume on the absorbance of the ion-pair complexes of Colistin (3 µg mL− 1) and Polymyxin B (5 µg mL− 1)

the most stable complexes with the best values of ABS.
All details have been illustrated in (Fig. 6).
Reaction time and temperature optimization

Based on the screening of color intensity that stayedstable for more than one day, the influence of time andtemperature on the yielded complex was investigated.
Reaction time was tested up to 30 min, and a slightincrease in ABS was noticed. Belongs to temperature,
turbidity was induced at the elevated degree of temperature that resulted in a decrease in ABS values. Consequently, 10 min at room temperature was the optimum
condition yielding the maximum ABS, as was observed in(Fig. 7).

Experimental solvent adjustment

To demonstrate how different solvents affected the ABS
values, numerous solvents including DW, acetonitrile,
acetone, methanol, and ethanol were investigated. The
best outcomes were obtained by employing DW as thediluting solvent that reflects the greenness of the presented work. All data were displayed in (Fig. 8).
Stoichiometric mole fraction method

Equimolar (3 × 10− 4 M) standard solutions of CS, Poly
B, and EB were used to implement Job’s method of continuous variations. The total volume of a series of complementary solutions was preserved at 1 mL. While theratios of the examined drugs and EB were created asfollows: (0.1:0.9, 0.2:0.8, 0.3:0.7, 0.4:0.6, 0.5:0.5, 0.6:0.4,

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Fig. 7 Effect of time on the absorbance of the ion-pair complexes of Colistin (3 µg mL− 1) and Polymyxin B (5 µg mL− 1)

Fig. 8 Effect of diluting solvent on the absorbance of the ion-pair complexes of Colistin (3 µg mL− 1) and Polymyxin B (5 µg mL− 1)

0.7:0.3, 0.8:0.2, and 0.9:0.1), then the general analyticalmethodology was implemented. As a result, the mole
fraction of 0.5 outputted the highest results that revealed
the molar ratio between the studied drugs and EB dye
was 1:1. All details were illustrated in (Fig. 9).
Validation study

In agreement with ICH guidelines [51], the validity ofseveral analytical parameters was examined. Linearity and range, the limit of detection (LOD), the limit ofquantification (LOQ), precision, accuracy, and robustness were examples of these screened factors.
Range of Linearity

By drawing the obtained absorbance against CS and
Poly B at different concentrations (µg mL− 1), calibration

curves were constructed. In the range of 1–6 and 1–9 µg
mL− 1 for CS and Poly B, respectively, the relationshipbetween the concentrations and the absorbance values
was linear as indicated by high coorelation coefficientvalues (≥ 0.9990) and low standard deviation of residuals
from line valus. All analytical parameters of both studied
drugs that belong to the linear regression equation weregathered in (Table 1).
LOD & LOQ

LOD and \ LOQ were calculated to assess the sensitivity
through the formulas 3.3 ϭ /S and 10 ϭ/S, respectively.
Where ϭ is the standard deviation of the intercept and S
is the calibration curve’s slope. The values for both citeddrugs were gathered in (Table 1).

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Fig. 9 Job’s technique for determining the stoichiometry of the reaction using 3 × 10− 4 M concentration of both erythrosine B and Polymyxins

Table 1 Spectral features and validation parameters for thestudied colorimetric approach
Parametersλεξ (νµ)Linear range (µg mL− 1)
Correlation coefficient (r)Determination coefficient (r2)Intercept ± SD*
Slope ± SD
Sy.x*****LOD (µg mL− 1)****LOQ (µg mL− 1)

Poly B545
1–9
0.99950.999

CS0.3882 ± 3.671 × 10− 3
0.0552 ± 6.52 × 10− 4
0.005053
0.22
0.66

0.3745 ± 2.449 × 10− 3
0.0534 ± 6.21 × 10− 4
0.00294
0.15
0.46

Precision

1–6
0.99950.9991

The acceptability criteria for linearity is expressed as (r) and should be ≥ 0.9990*SD: Standard Deviation ** Sy.x: Standard Deviation of residuals from lines***LOD: Limit of detection. ****LOQ: Limit of quantitation

2
4
6
Mean*
SD
RSD

Poly BFound
conc.
(µg mL− 1)
1.987
4.076
5.923

%
Recovery*
(%R)
99.35
101.90
98.72
99.99
1.68
1.68

Found
conc.
(µg mL− 1)
2.098
3.929
6.055

CS
%
Recovery
(%R)
98.60
98.23
100.92
99.25
1.46
1.47

% R: should be in the range (98–102%) * mean: of three replicate measurements

Accuracy

The closeness of the experimental values to each otherwas assessed in two ways. Firstly, intra-day precision, by
replicating analysis of three different concentrations (2,4, 6 µg mL− 1) of both experimented drugs at three different times along the day. Secondly, inter-day precision,by checking the same concentrations through successive
three days. The calculated relative standard deviation(RSD) does not exceed 2, which is the accepted criteria,
confirming the good precision of the described work. Alldata were illustrated in (Table 3).
Robustness

Table 2 Evaluation of the accuracy of the described
spectrophotometric approach
Taken conc.
(µg mL− 1)

in its acceptable range (98–102%), proving the accuracyof the proposed method, as shown in (Table 2).

Three drug concentration levels (2, 4, and 6 µg mL− 1)
were examined to evaluate the suggested method’s accuracy. The calculated percentage recovery (%R) showed agood agreement between the measured and actual values

The method’s capacity to remain unaffected by small
intentional changes to the experimental parameters without significant alterations to measure the absorbancevalues is referred to as robustness. Four factors from theexperiment were screened: pH, buffer volume, dye volume, and reaction time. It was revealed that none of thesefactors has significantly affected. Thus, the establishedspectrophotometric approach was said to be robust andcan be routinely employed in the assay of studied drugs.
All outcomes were displayed in (Table 4).
Applications of the developed approach
Assay of PMS in parenteral and oral dosage forms

Quantification of intravenous dosage forms of PMS
and oral Colistin sulfate® tablets was successfully implemented. Through statistically comparing the outcomeswith the previously reported article [10], we used the Student’s t-test and the F-test to evaluate the precision andaccuracy. Consequently, there was no major differenceAbdelmajed et al. BMC Chemistry(2024) 18:158

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Table 3 Intra- and inter-day precisions evaluation for PMS analysis
Concentration (µg mL− 1)2
4
6

Poly B%Mean Recovery* ± RSD
Intra-day
99.76 ± 0.88
100.13 ± 1.24
98.44 ± 1.77

CSInter-day
98.47 ± 1.12
99.22 ± 0.69
100.62 ± 0.38

Intra-day
101.61 ± 0.77
99.51 ± 1.48
98.16 ± 0.94

Inter-day
99.53 ± 0.72
100.65 ± 1.74
99.20 ± 0.42

RSD: relative standard deviation (should not exceed 2 ) * Mean of three determinations

Table 4 Robustness for determination of poly B and CS by thedeveloped approach
Method Parameters
pH
3.8
4
4.2
Buffer volume
0.8
1
1.2
Volume of erythrosine B
0.4
0.5
0.6
Time of reaction (min)5
1015Poly B%R* ± SDCS

98.32 ± 1.87
99.88 ± 0.91
100.51 ± 1.65

99.93 ± 0.44
101.54 ± 1.76
98.74 ± 1.58

1.01.32 ± 0.99
99.14 ± 0.87
98.87 ± 1.36

99.05 ± 0.92
100.04 ± 1.36
98.11 ± 1.51

100.92 ± 0.80
98.70 ± 1.35
101.81 ± 1.79

98.80 ± 1.27
99.09 ± 0.94
101.71 ± 1.55

101.44 ± 1.93
99.71 ± 0.76
97.99 ± 1.63

99.11 ± 1.59
99.84 ± 0.52
99.48 ± 0.99

% R: should be in the range (98–102%) * Mean of three determinations

Table 5 Screening of PMS parenteral dosage forms by
developed approach and compared with the reference approach
Marketform
Colomycin® vial
Paximid® vial

Developed
method%R ± SD*
100.05 ± 0.66
99.79 ± 1.04

Reference
method99.54 ± 1.57
99.62 ± 0.39

t-value**
0.851
0.468

f-value
2.233
1.477

*

Mean is the average of five determinations. ** Tabulated values at 95%
confidence limit are t value = 2.306, F value = 6.338

between the suggested approach and the published one.
All values were inserted in (Table 5).
Test of Content Homogeneity

The tested tablets exhibited the acceptable uniformity ofCS, as shown by the estimated acceptance value (AV),which was 3.456 and was less than the highest permissible acceptable value of 15. All information was gatheredin (Table 6).

Greenness assessment
Many tools are now available to assess and contrast
the greenness of different analytical techniques. GAPI(Green Analytical Procedure Index) tool has the unique

Table 6 Content uniformity of CS in tablet form
Tablet number

1
2
3
4
5
6
7
8
9
10
Mean
SD
RSD
Acceptance value (AV)Maximum allowed AV (L1)*

Mean % R* of
the claimed
content Colistin
sulfate ® tablet
98.78
99.51
97.41
101.33100.27
98.94
96.99
98.77
99.82
101.22
99.30
1.44
1.45
3.456
15

Mean is the average of three determinations

advantage of covering the entire analytical method [52]. It
consists of five pentagrams, each of which denotes a stepin the analytical technique: sample preparation and collection; chemicals and solvents; applied equipment; andthe goal of the analytical technique. Three color codes
are used by GAPI: red denotes a significant environmental threat, while yellow and green denote a lower risk andincreased greenness. AGREE (the Analytical GREEness)is also a flexible, and straightforward assessment tool thatprovides an easily interpretable and informative result[53]. The assessment criteria are taken from the 12 principles of green analytical chemistry and are transformed
into a number. The result is a pictogram indicating thefinal score performance of the analytical procedure ineach criterion and weights assigned by the user. All data
were inserted in (Figs. 10 and 11). The good G score, 0.73,
conflicted high greenness of the current work owing
to utilizing distilled water throughout the procedures
without extraction process and drastic conditions likeheating [13, 14] or boiling [9, 11] or employing organic
solvent [10], this is in light of comparison with the otherreported fluorimetric techniques. On the other hand,
not consuming large amounts of energy as in LC-MS
articles or huge volumes of organic solvents like in HPLC

Abdelmajed et al. BMC Chemistry(2024) 18:158

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for the routine assay of the studied drugs instead of thepreviously published spectroscopic and chromatographic
techniques in quality control laboratories. Moreover,Content uniformity testing for CS tablets was employed
and the described current work was considered the firstcolorimetric method employed to quantify the intravenous market forms of the cited drug.
Author contributions
Mahmoud A. Abdelmajed: Methodology and writing the original draft, Khalid
M. Badr El-Din: Validation and Review, Tamer Z. Attia: reviewing and publishingediting; Mahmoud A. Omar: reviewing.

Fig. 10 Pictogram of the developed method using GAPI tool

published papers for the cited drugs, all previously mentioned factors negatively affected the G score. Recently,
more than one method was employed for the assessmentof the methodology such as cGAPI, AGREEprep, The upto-date ChlorTox Scale indicator, CALIFICAMET tool,
RGB12 algorithm, and spider diagram [54–57].

Conclusion
The screened medications were quantified by employing
a green, easy-to-implement, and economical spectrophotometric approach. The produced complexes werewater-soluble, therefore there was no need for organic
solvents, making the methodology safer for the environment. Furthermore, the suggested approach is more
efficient in terms of time reliability, and sensitivity thanother reported spectrophotometric methods. In light of
this, the described approach can be easily implemented

Fig. 11 Pictogram of the developed method using AGREE toolFunding
Non-funding.
Open access funding provided by The Science, Technology & Innovation
Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank
(EKB).
Data availability
No datasets were generated or analysed during the current study.

Declarations
Ethics approval and consent to participate
Guidelines according to BMC journal.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Received: 4 March 2024 / Accepted: 5 August 2024

Abdelmajed et al. BMC Chemistry(2024) 18:158

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