(2024) 18:118
Abdullatif et al. BMC Chemistry
https://doi.org/10.1186/s13065-024-01220-4

BMC Chemistry

RESEARCH

Open AccessUtilizing two different sustainable and green
spectrophotometric approaches using
derivative ratio spectra for the determinationof a ternary severely overlapped mixture:application to veterinary formulation
Hind A. Abdullatif1*, Mohammed Abdelkawy2*, Maha Kamal1 and Nesma M. Fahmy1

Abstract
Mathematical manipulation technique has proven to be a very powerful tool for efficient processing and handlingof highly overlapped spectra. This work introduced two green and sustainable approaches for the successful recoveryof the ternary mixture, Tylosin tartarate (TYL), Sulfadimdine (SLD), and Trimethoprim (TRI). The approaches wereconstructed using three different methods, derivative ratio spectrum zero-crossing method (DRSZ), double divisor
ratio spectra derivative method (DDRD), and factorized derivative ratio method coupled with spectrum subtraction(FDRM-SS). The two approaches succeeded in recovering the three drugs (Linearity range achieved were 0.5–5 µg/
mL for TYL, 0.3–1.3 µg/mL for SLD, and 0.3–5 µg/mL for TRI), giving convenient standard deviations and satisfactory
recovery percentages. The recommended methods have been verified in accordance with (ICH) guidelines. Whenthe results were statistically compared to the official methods, no significant difference was found. Both AGREE—
Analytical GREEnness Metric Approach and Software, and White Analytical Chemistry (WAC) RGB model gave scoresof 0.93 and 97.2%, respectively, which proved that the approaches were eco-friendly and abiding by the sustainability
principles.
Keywords White analytical chemistry, Analytical GREEnness Metric Approach and Software, Veterinary, Sustainable,
Tylosin, Sulfadmidine, Trimethoprim, Ternary severely overlapped*Correspondence:
Hind A. Abdullatif
hanoood7@hotmail.com
Mohammed Abdelkawy
dr.mohabdelkawy@hotmail.com
1
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ahram
Canadian University, Cairo, Egypt
2
Analytical Chemistry Department, Faculty of Pharmacy, Cairo University,
Giza, Egypt

Introduction
In recent decades, animal health has received
considerable attention due to its crucial importanceand contribution to the human dietary system and wellbeing. Thus, to increase animals’ lifetime and wellness,light was spotted on veterinary antibiotics, and the safety
and efficacy of them. Consequently, various antibiotic
formulations have been recently fed to the market all ofwhich are of great impact to animal’s welfare. Therefore,
the quality control of veterinary formulations has been
subjected to numerous controls and regulations, and any© 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 theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​
mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Abdullatif et al. BMC Chemistry(2024) 18:118

study on this very important segment of veterinary drugs
is considered significant and lifesaving.
Tylosin tartrate(TYL), having IUPAC name of(4R ,5S,6S,7R ,9R ,11E,13E,15R ,16R)-15-[[(6-deoxy2,3-di-O-methyl-b-d-allopyranosyl)oxy]methyl]-6[[3,6-dideoxy-4-O-(2,6-dideoxy-3-C-methyl-a-l-ribohexopyranosyl)-3-(dimethylamino)-b-d-glucopyranosyl]oxy]-16-ethyl-4-hydroxy-5,9,13-trimethyl-7-(2-oxoethyl)oxacyclohexadeca-11,13-diene-2,10-dione, as shownin Fig. 1. It is a macrolide antibiotic, Considered a very
important antibacterial agent in veterinary formulations[1]. TYL possesses bacteriostatic action against bacteria and Gram-positive mycoplasma [2]. Additionally, it
is added to poultry feed as an anticoccidial ingredient.
Different methods were described in literature for TYL
determination depending on UV–VIS spectrophotometry [3–5], chromatographic methods [6, 7], and electrochemical methods [8, 9].
Sulfadimidine (SLD), having IUPAC name of
4-amino-N-(4,6-dimethylpyrimidin-2-yl)
benzenesulfonamide, is shown in Fig. 1. It is a sulfa-based drug,
used in hindrance and management of various bacterial
invasions as actinomyces, and urinary tract infections[10]. Sulfonamides are commonly used in veterinary
practice. Several methods of determination werementioned in literature, including Spectrophotometricmethods [11, 12], chromatographic methods [13–15],and electrochemical methods [16].
Trimethoprim (TRI), having IUPAC name of5-(3,4,5-Trimethoxybenzyl)
pyrimidine-2,4-diamine,is shown in Fig. 1. It is a well-known potent metabolic
inhibitor of dihydrofolic acid reductase [17]. This drug
shows powerful antibacterial activity against strains
which are resistant to other frequently used antibiotics,
e.g., β-lactams [18].
The three drugs, TYL, SLD, and TRI are found as aternary mixture in the Egyptian market under the name
Tylosin ­Plus®, and veterinarians rely on this formulation
regularly for the treatment of various bacterial invasions

Fig. 1 Chemical structures of the three cited drugs

Page 2 of 15

in large animals. The three drugs are also mentioned in
the British Pharmacopoeia [19–21].
As far as we know, there was a chromatographic
method in literature describing the determination of
the ternary mixture [22–24]. Unfortunately, HPLC wasproved to be time-consuming and of high cost, with a
lot of chemicals used and waste produced. There is noother work in literature describing an easier and simple,
yet green method, like spectrophotometry, for thedetermination of ternary mixture.
Unfortunately, multicomponent mixtures, including
this studied mixture, typically have highly overlapping
spectra, which urged the buildup of various methods
for determining each studied drug’s concentration [25].
Recent incorporated spectrophotometer software that
supports mathematical techniques is believed to be an
optimal key to resolve complicated spectra, and to get
as much analytical data as possible from the unresolved
bands [26].
Thus, this study investigated the applicability of using
green spectrophotometric methods as; Derivative ratio
spectrum zero-crossing method (DRSZ), Double divisor
ratio spectra derivative method (DDRD), and Factorized
derivative ratio method coupled with spectrum
subtraction (FDRM-SS); which are able to resolve
severely overlapped ternary mixtures.
Additionally, we aimed to magnify green and white
chemistry recognition; we took into consideration
sustainability precautions and guidelines throughout the
study. Sustainability is a major focus of green chemistry,
which seeks to improve resource efficiency and lessen
its negative effects on the environment. Specifically, thereduction or elimination of hazardous chemicals is given
priority in green analytical procedures. In addition to
improving worker safety in the laboratory and aim to
reduce waste and pollutant production. Adopting green
analytical techniques encourages innovation in techniquedevelopment, complies with regulatory requirements,
and responds to public concerns about environmental

Abdullatif et al. BMC Chemistry(2024) 18:118

responsibility. Green analytical methods incorporate
energy efficiency and renewable resource principles,
which lead to more conscientious laboratory practices. In
the end, this strategy contributes positively to the larger
objective of sustainable scientific research [27–29]. The
methods’ greenness was evaluated with AGREE greenness
tool, where each sector of the clockwise figure illustrating
the 12 principles is colored from red to yellow to green,signifying how green each principle is in the methodology.
The score is usually closer to one the greener the diagram.
The whiteness was assessed by RGB 12 model, where
along with the environmental factors, WAC considers
two more critical standards that impact the method’s
quality: analytical (red) and practical (blue). An analysisof whiteness using the RGB color model, which states thatthe combination of red, green, and blue light beams creates
the illusion of whiteness, demonstrates the coherence andsynergy of the analytical, ecological, and practical features.

Materials and methods
Theoretical background
Derivative ratio spectrum zero‑crossing method (DRSZ)

In this method [26], a combination containing X, Y andZ that show a severely overlapped spectra, is divided by
one of the drugs’ standard solutions, and the DD1 of theresulted spectrum is found (the divisor component isremoved by derivatization). To find the concentration of
one component at the other component’s zero-crossing
point, modification is done in the matching regression
equation by changing the peak amplitude.
Double divisor ratio spectra derivative method (DDRD)

The three-component mixture (X + Y + Z) is dividedby (X + Y); an equimolar mixture, then followed byderivatization [26]. In this case, Cz concentration is directly
proportional to the derivative at the coincidence points,
which represent the minimum and maximum wavelengthfor pure Z, and Z in the ternary mixture (X + Y + Z).
Similar methods could be utilized in estimation of C
­ x and­CY in the ternary combination.
Factorized derivative ratio method coupled with spectrum
subtraction (FDRM‑SS)

This method [30] is a methodology for factorized spectra[31–33] to resolve severely overlapped ternary mixtures
spectra (X + Y + Z). In this method, divisor (Z’)’s
contribution is negated by derivatization utilizing anappropriate sequence, so Z/Z’ could be negated.
To apply this, an appropriate wavelength is chosen, in
which X/Z’ spectrum happens to own a contribution at
the zero crossing point of the third drug Y/Z’ (could be

Page 3 of 15

zero-crossing or zero-contribution point) to obtain X
factorized derivative ratio, in which ­Px(λzero point)=1, ­DD1of X is divided by its recorded peak amplitude value in thisspecific ­wavelength(λzero point).

DD1 factorized spectrum (FS ′ ) = PX /PX V( zero point) .
The component Z’ is used as a divisor to analyze
laboratory-prepared mixtures, where the recorded
amplitude is multiplied at a certain wavelength (λ zero point)by the formerly attained X ­DD1 factorized spectrum,
derivatization under specified experimental parameter,the ­DD1 spectrum of the elements X(X/Z’) could befound [29].
Instrumentation

Spectrophotometric measurements were done withthe help of an ACER compatible computer that was
associated with double beam UV-VIS spectrophotometermodel J-760 (Jasco, Japan), using 1.00 cm quartz cells.
The Scans were completed between the range of 200–
400nm at 0.1 nm intervals, using S­pectraManager®
software. Sonication was done by an ultra-sonicator(Crest Ultrasonics Corp., NJ, USA).
Chemicals and reagents(a) Pure samples: SLD, TYL, and TRI with purity
100.06% ± 0.637, 100.45% ± 0.50 and 100.09%± 0.583, respectively bestowing to the BP official
methods, were generously provided by Pharma­Swede® Veterinary Company, Cairo Egypt.
(b) Veterinary formulation: Tylosin P­
lus® waspurchased from a veterinary drugstore in Egypt.
(c) Reagents: From (Sigma A­ldrich®, Germany),
analytical grade methanol was purchased.
Standard solutions

For the preparation of the standard solutions, 10 mg ofeach component were sonicated in 50 mL methanol
for 2 min separately, then each standard solution was
filled till 100 mL with methanol to prepare 100 µg/mL,
and TYL flask was enfolded with aluminium foil to be
sheltered from light.
Each drug’s working solution was prepared by adding
methanol till mark to a 10 mL volumetric flask that had
been filled with various concentrations of the standard
solution.

Abdullatif et al. BMC Chemistry(2024) 18:118

ProcedureSpectral data for TYL, SLD, and TRI

At 200–400 nm, the D­ 0 absorption spectra of the threecomponents were scanned against blank (methanol).
Factorized spectrum Using 1 µg/mL SLD as a divisor,
the TRI ­DD1 spectrum was divided by the amplitudes
obtained at 256 nm. Similarly, SLD ­DD1 spectrum using
1 µg/mL TRI was divided by the recorded amplitudes at
225.1 nm. The computer retained both output spectra.
Linearity and calibration graph construction Aliquots
from the 100 µg/mL standard solution equal to 0.3–1.3 µg/
mL SLD, 0.5–5 µg/mL TYL, and 0.3–5 µg/mL TRI weretaken to 10-mL volumetric flask. Each prepared standard
solution was scanned against blank (methanol) at a rangeof 200-400nm, which were retained on the computer.
Calibration graph of TYLApproach 1: DD1. ­D0 absorption spectra of TYL are
divided by 1 µg/mL SLD and plotting against readings at­Pmax 240 nm, then getting the ­DD1 spectrum.
Approach 2: DD1. ­D0 absorption spectra of TYL are
divided by 1 µg/mL TRI and plotting against readings at­Pmax 254.7nm then getting the ­DD1 spectrum.
Calibration graph of SLD
Approach 1: DRSZ: ­D0 absorption spectra of SLD was
divided once by 4 µg/mL TYL and its derivative wasobtained, and regression equation was constructed byplotting against 272.2 nm.
Approach 2: FDRM-SS: Using the divisor 1 µg/mL
TRI, the obtained spectrum represented SLD ­DD1. Then
to obtain a regression equation, the TRI concentrations
were plotted against the peak amplitude at 255.1 nm.
Calibration graph of TRI
Approach 1: DD1. ­D0 spectra were divided by 1 µg/
mL SLD, and first derivative was obtained, then plottedagainst readings at ­Pmax 245 nm.
Approach 2: DDRD. The ternary mixture was scanned,
along with the solution’ absorption spectra (different
TRI concentrations), then by the sum of 1 µg/mL TYL
and 1µg/mL SLD absorption spectra (double divisor) toobtain the ratio spectra. Afterwards, their derivatives
were obtained, and used for the construction of theregression equation, where the peak amplitude attheir coincidence point (236.5nm) was plotted againstconcentrations of TRI.
Validation procedures

Linearity and range: the proposed methods’ linearity
range was valued by checking the concentrations of all
three drugs, three times each, and inspecting whichrange gave best sensitivity.

Page 4 of 15

Accuracy: For investigating the proposed method’s
accuracy, various concentrations found in thelinearity range of each drug were repeated three times
implementing the proposed methods.
Precision: Validating repeatability and intermediate
precision is crucial. This was done by examining 3
separate concentrations of every studied component
within its linearity range. This was done by analyzing 0.6,
0.8 and1.0 µg/mL for each drug three times, on a single
day, to obtain repeatability. While concentrations 0.5,
0.7 and 0.9 µg/mL for SLD, 1, 3 and 5 µg/mL for TRI and
TYL, where investigated on three consecutive days toobtain intermediate precision.
Specificity: The proposed methods’ specificity was
ensured by inspecting multiple mixtures of (SLD+TRI+
TYL) within the linearity ranges using diverse ratios.
Concentrations used for this parameter were mixturesof 2.00, 1.00 and 2.00 µg/mL, 3.00, 1.00 and 3.00 µg/mL,
4.00, 1.00 and 4.00 µg/mL, 5.00, 1.00 and 4.00 µg/mL,
and 1.00, 1.25 and 0.50 µg/mL for TYL, SLD, and TRI
respectively.
LOD and LOQ: The standard deviations of theregression line residuals and the calibration curve’s slope
were used to calculate LOD and LOQ, then substituting
them respectively in equations:

LOD =(3.3)(SD).
(slope)

(1)

LOQ =

(10)(SD).
(slope)(2)

Applying the proposed spectrophotometric methods fordetermining SLD, TYL, and TRI in laboratory-preparedmixturesApproach 1: DRSZ method was applied to obtain SLD
concentrations, followed by applying FDRM-SS to obtain
TRI and TYL concentrations in the ternary mixture as
follows:
After obtaining the ternary mixture’s ­DD1 ratio spectra
and using 4 µg/mL TYL as a divisor, measurements at
272.2 nm were substituted in the matching regression
equation to get the SLD concentrations.
The ternary mixture’s ­DD1 ratio spectra were obtained,
and measurements at 272.2 nm were inserted in thematching regression equation to obtain the SLD
concentrations. 4 µg/mL TYL was used as a divisor.
Using 1 µg/mL SLD as a divisor, the ­DD1 of the ternary
mixture’s ratio spectrum was found. By multiplying
the peak amplitude at 256 nm by the previously
saved factorized TRI spectrum, the ­DD1 TRI alone
was resolved. The peak amplitude at 245 nm fromthe resolved ­DD1 TRI was substituted in the relevantAbdullatif et al. BMC Chemistry(2024) 18:118

Page 5 of 15

regression equation to calculate the concentrationof TRI. Ultimately, ­DD1 TYL alone is obtained bysubtracting the derived ­DD1 TRI spectrum from the1
ternary mixture’s ­DD spectrum. The 240 nm readings inthis spectrum were replaced into its matching regression
equation to determine the amount of TYL present in the
combination.
Approach 2: DDRD was utilized to obtain SLD and TRI,
then FDRM-SS was applied to obtain TYL concentrationsin the ternary mixture as follows:An equimolar double divisor (1 µg/mL TYL + 1µg/
mL SLD) was used to divide the ternary mixture, andthe resulting spectra were then derivatized. The TRI
concentration was obtained by substituting readings at
236.5nm in its corresponding regression equations.
To resolve D­ D1 of SLD alone, the first derivative ratio
spectrum of the ternary mixture was divided by 1 µg/
mL TRI, and its peak amplitude at 225.1 nm was thenmultiplied by the previously computed SLD factorized
spectrum. The peak amplitude at ­Pmin (P 255.1) was
inserted in the corresponding regression equation tofind the concentration of SLD. Lastly, ­DD1 TYL alone
is obtained by performing spectrum subtraction,
which subtracts the resulting D­ D1 SLD from the1
ternary mixture’s ­DD spectrum. The concentrationof TYL in the mixture was determined by inserting
readings at 254.7 nm in this spectrum in the associatedregression equation.

One mL of Tylosin P­ lus® contains 100 mg TYL, 125
mg SLD, and 25 mg TRI, and 25 mg of TRI were added
as a standard addition. Firstly, 1 mL of the dosage
form was transferred into a 100 mL volumetric flask
and completed with methanol till mark. From this, 1
mL is taken in another 100 mL volumetric flask andcompleted till mark. Finally, 1 mL was taken into a
10 mL volumetric flask and fulfilled with methanol,
obtaining a final concentration of 1: 1.25: 0.5 µg/mL of
TYL, SLD, and TRI. To obtain the true value of TRI,
0.25 µg/mL of the standard addition was subtracted.
Prepared flask was stored away from light.
Application to the veterinary formulation

Results
Both approaches gave convenient and satisfactory results
and recoveries. Regarding Approach 1, TYL accuracy was
100.29, and standard deviation was 0.741. As for SLD,
accuracy and standard deviation were 100.77 and 0.179,
respectively. Finally, TRI owned accuracy and standard
deviation of 100.78 and 0.650.
As for Approach 2, TYL gave results of 100.68 and 1.05
regarding accuracy and standard deviation (standard
deviation was higher than that of Approach 1). While
SLD gave 100.41 and 0.518 for both results. Finally, TRI’s
results were 100.27 and 0.391.
Linearity ranges were 0.5–5 µg/mL, 0.3–1.3 µg/mL,
and 0.3–5 µg/ mL for TYL, SLD, and TRI, respectively.
It was clear that Approach 1, where DRSZ, FDRM-SS
was utilized, showed lower manipulation steps. Approach
2 had certain limitations as DDRD method requires

Table 1 Assay parameters and method validation obtained by applying the proposed spectrophotometric methods
MethodTYL
1

DD(TYL/SLD) (240 nm)
Approach 1

SLD
1

1

DD
(TYL/TRI) (254.7 nm)
Approach 2

DD(272.2 nm)
Approach 1

TRI
1

DD
(255.1 nm)
Approach 2

DD1(245 nm)Approach 1

0.3–1.3

DDRD(236.5 nm)
Approach 2

Range (µg/mL)

0.5–5.0Slope

0.00640.0981

0.05360.01

0.1566

0.3–5.0
0.0098Intercept

Correlation ­coefficient®

0.00050.0073− 0.006− 0.0020.00020.00130.99980.99980.99991.00000.99980.9998Accuracy

100.68100.29100.77100.50100.78100.27

Repeatability (RSD)a

0.089

0.060

0.627

1.425

0.517

1.526

Intermediate Precision (RSD)b

0.713

0.562

0.976

0.750

0.880

0.822

Specificity
Mean ± SD

100.85 ± 0.800

100.29 ± 0.740

100.77 ± 0.179

100.28 ± 1.3

99.82 ± 1.223

100.49 ± 1.052

LOQ (µg/mL)

0.254

0.500

0.279

0.260

0.057

0.300

LOD(µg/mL)

0.084

0.166

0.093

0.085

0.019

0.100

a

RSD relative standard deviation of 0.6, 0.8, 1.0 µg/mL for the three drugs were usedb

RSD (n = 3) relative standard deviation of concentrations 0.5, 0.7, 0.9 µg/mL for SLD, 1, 3, 5 µg/mL for TRI and TYL

Abdullatif et al. BMC Chemistry(2024) 18:118

Page 6 of 15

equimolar divisor concentrations and a coincidence
point where measurements are conducted.
Validation

Method validation was fulfilled with respect toInternational Conference of Harmonization (ICH)
guidelines [30].

99.82 were the results of TRI. So, we could say that successful and satisfactory recoveries were obtained, as
shown in Table 1.
Limit of detection and limit of quantitation (LOD and LOQ)

Concentrations of the drugs were found to be linear
at ranges 0.3–5 µg/mL, 0.3–1.3 µg/mL, 0.5–5 µg/mL
regarding TRI, SLD, and TYL, respectively in methanol.
The range was satisfactory and allowed manipulation at awide range, as shown in Table 1.

Usually, acceptable results of LOD and LOQ areassociated with 95% probability of obtaining a correct
result [34]. Satisfactory results were shown, considering
LOQ, 0.500 and 0.254 for TYL, 0.279, 0.050, and 0.260
regarding SLD, and 0.057 and 0.300 regarding TRI.
As for LOD, 0.166 and 0.084 were TYL results, 0.093,
0.016, and 0.085 were SLD’s findings, and finally 0.019
and 0.100 were TRI’ results. Both LOD and LOQ results
were shown in Table 1.

Accuracy

Application to the veterinary formulation

Linearity and range

The accuracy readings were 100.68 and 100.29 regarding
TYL, while 100.77, 100.41 and 100.50 for the threemethods applied to SLD, and finally 100.27 and 100.78
regarding both methods applied to TRI. As expressedin Table 1, Concentrations were attained with favorable
percentage recoveries lying within the accepted range.
Precision

Precision plays a pivotal role in ensuring validity andreliability, as it is crucial to ensure that the methods
proposed produce consistent results each time they areapplied. Speaking of repeatability, results were 0.089 and
0.060 regarding TYL, 0.627, 1.663 and 1.425 regarding
SLD, and 1.526 and 1.517 regarding TRI. The results weresatisfactory, as clarified in Table 1.
Specificity

Another validation factor examined was specificity.
Results of TYL were 100.85 and 100.29 obtained fromthe two methods applied, while 100.77, 99.71, and 100.28
were the specificity results of SLD. Finally, 100.49 and

The formulation’s three components, TYL, SLD, and TRI
were found in a challenging ratio, 1:1.25:0.25. Therefore,
it was not possible to identify the minor component
TRI concurrently with the other two components,
considering the linearity range of each component.
Unless enrichment technique; standard addition [35],
was applied. Minor component (TRI) was enriched bystandard addition of exactly known concentration of TRI,
so that the total response of the veterinary formulation
could be successfully replaced in the regression equation
to get the accurate concentration of TRI. Subtracting thequantity of standard addition added (amount equivalent
to 0.25 µg/mL) from the total drug concentration in theanalyzed sample yields the actual TRI concentration
present in the dose form.
Table 2 reveals acceptable recovery percentages
when applying the proposed methods to the veterinary
formulations. Recovery findings like 100.78, 100.74, and101.90 for TYL, SLD, and TRI, respectively were obtainedby the various methods of both approaches. This ensured
the success of the proposed methods.

Table 2 Analysis of TRI, SLD, and TYL in Tylosin ­Plus® injection
Tylosin Plus
Batch No. ­5020118®
Drug

TYL
1

MethodDD(TYL/SLD) (240 nm)
Approach 1

Claimed Conc

1 µg/mL

b

SLD
1

DD
(TYL/TRI)(254.7 nm)
Approach 2

1

DD(272.2 nm)
Approach 1

TRI
1

DD
(225.1 nm)
Approach 2

DD1(245 nm)Approach 1

DDRD(236.5 nm)
Approach 2

0.25 µg/mLa

1.25 µg/mL

Found%

100.78100.44100.74100.40101.91102.00± S.D± 0.800± 1.120± 0.177± 0.560± 1.110± 1.050aAfter subtraction of 0.25 µg/mL TRI standard additionb

All results were the average of three different readings

Abdullatif et al. BMC Chemistry(2024) 18:118

Page 7 of 15

Table 3 Statistical comparison between results obtained by the proposed methods and the official methods [20–22]
Drug

TYL

SLD

MethodDD1(TYL/SLD)(240 nm)Approach 1

DD1(TYL/TRI)(254.7)
Approach 2

Mean

TRI

Official­methoda

DD1(272.2 nm)
Approach 1

DD1(255.1 nm)
Approach 2

Official
method b

DD1(245 nm)Approach 1

DDRD(236.5 nm)
Approach 2

Official­methodc

100.51

99.82

100.4599.93

100.23100.06100.0499.88

100.09

S.D.

1.051

0.727

0.500

0.521

0.302

0.637

0.744

0.766

0.583

Variance

1.10400.

0.52850.25000.27140.09120.40570.55350.58670.3398F test(5.05)

4.416

2.112

–

1.498

4.448

–

1.628

1.726

–

Student’s
t-test(1.81)

0.13

1.7

–

0.401

0.587

–

0.110

0.514

–

The figures in parenthesis are the corresponding theoretical values at P = 0.05
The sample number(N) was 5 for all methodsaBP Official method includes HPLC method using C18 column and sodium perchlorate (pH 2.5) adjusted with 1 M hydrochloric acid: acetonitrile (60,40, v/v) as mobile
phase

b

BP Official method includes potentiometric nitrite titrationc

BP Official method includes HPLC method using methanol R and sodium perchlorate in ratios (30:70v/v) adjusted to pH 3.6 using phosphoric acid

Table 4 One-way ANOVA testing for the different proposed
methods compared to the official methods [20–22] for thedetermination of TYL, SLD, TRI in pure powdered form
Source of variation

SS

df

MS

F

F crit

1.730

2

0.865

1.378

3.682

Within groups

9.413

15

0.627

Total

11.14417

Between groups

0.136

2

0.068

Within groups

7.400

15

0.493

Total

7.536

17

Between groups

0.278

2

0.139

Within groups

3.842

15

0.256

Total

4.120

17

TYL
Between groups

Table 5 One-way ANOVA testing for the dosage form recovery
compared to the method reported in literature [20–22]
Source of variation

SS

df

MS

F

F crit

3.970

5.143

1.089

5.143

0.265

5.143

TYL
Between groups

6.919

2

3.459

Within groups

5.227

6

0.871

Total

12.147

8

SLD

TRI
0.138

3.682

Between groups

2.299

2

1.149

Within groups

6.331

6

1.055

Total

8.631

8

TRI

SLD
0.543

3.682

Between groups

0.703

2

0.351

Within groups

7.958

6

1.326

Total

8.661

8

Statistical analysis

Statistical analysis was done by comparing the outcomes
found by the spectrophotometric methods proposed inthis study with the BP official methods [19–21]. After
undergoing an ANOVA, t-test, and f-test on students, thecomputed t and F values were lower than the theoreticalones. This showed that, in terms of accuracy and precision,
there was no discernible difference between the outcomesachieved and the official methods. Moreover, ANOVA wasalso used to compare the recovery claimed from the dosage
form using the proposed methods with the method foundin literature [19–21]. Both ANOVA results revealed no significant difference, which was illustrated in Tables 3 andFig. 2 AGREE software results for the proposed mixtureAbdullatif et al. BMC Chemistry(2024) 18:118

Page 8 of 15

Fig. 3 Visualization of the evaluation results according to the RGB method for the proposed approaches

4. Finally, One-way ANOVA testing for the dosage form
recovery was compared to the method reported in literature [20–22] (Table 5).
Greenness assessment

The AGREE score for this study’s proposed methods of
GAC was 0.93. The least score was given to point 2 and 5
in the AGREE assessment, referring to sample amount andautomation, as shown in Fig. 2. Likely, WAC evaluation
model had a score of 97.2, as shown in Fig. 3.

Discussion
Tylosin ­Plus® is a reliable antibiotic veterinary
formulation used for the treatment of various bacterial
invasions in large animals. Its crucial importance andreliance among veterinarians made it vital to further

Fig. 4 Overlayed Zero order absorption spectra of 1 µg/mL TYL (_._._.), SLD (___), and TRI. (. . .) in methanol

Abdullatif et al. BMC Chemistry(2024) 18:118

investigate its method of analysis and develop a reliable
and easily applicable methodology for its determination.
The first challenge found in this combination was thatthe three drugs showed a severely overlapped zero order
spectra in the region 200–330nm, as shown in Fig. 4.
There were two approaches demonstrated in this study
that successfully recovered all the three drugs in thiscomplex overlapped mixture illustrated in Fig. 1. Thiswas done by utilizing DRSZ, DDRD, and FDRM-SS.
Approach 1: Figure 5 illustrates the whole approach.
By getting the ternary mixture’s ­DD1 ratio spectra (SLD+ TRI+ TYL) utilizing TYL as a divisor, contribution of
TYL in the mixture was cancelled leaving only contribution due to TRI and SLD. SLD was successfully recovered

Page 9 of 15

by derivative ratio zero crossing point (DRSZ). As shownin Fig. 6, the SLD and TRI ratio spectra’s first derivative showed a peak at 272.2 nm, where TRI has zero
contribution.
Substitution of the peak amplitude at 272.2 nm in thecorresponding regression equation, resulted in SLD
concentrations alone in the ternary mixture. Several
attempts for applying DRSZ to obtain TRI or TYL,
but all came to failure, as there was no peak amplitude
where the other drug had zero contribution. So, anothermethod was applied to continue resolving TRI and TYL
which is FDRM-SS.
As the concentration of SLD was successfully obtainedin the mixture, 1 µg/mL SLD was selected as a divisor for

Fig. 5 Scheme for approach 1

Fig. 6 First derivative absorption spectrum of 1 µg/mL of TRI and SLD using 4 µg/mL TYL as a divisor

Abdullatif et al. BMC Chemistry(2024) 18:118

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Fig. 7 First derivative spectra of both 1 µg/mL TRI and TYL using 1 µg/mL SLD as a divisor

the ternary mixture. Upon derivatization, SLD contribution was cancelled leaving the binary mixture of ­DD1 TRI
and ­DD1 TYL. There was no peak amplitude showing
zero contribution of the other drug but luckily, there wasan amplitude at 256 nm where TRI had a contribution atzero crossing point of TYL, as shown in Fig. 7, therefore
FDRM-SS was successfully applied.
Amplitude at 256 nm were multiplied by thefactorized spectrum of TRI constructed using thispeak amplitude (a/a 256 nm). The resulted spectrum
represented ­DD1 TRI alone, and readings at 245 nm
were replaced in its corresponding regression equation.
Finally, spectrum subtraction (SS) was applied; after
subtracting the resulted ­DD1 TRI spectrum from ­DD1of the ternary mixture using 1 µg/mL SLD as a divisor,
the resulted spectrum should be ­DD1 TYL alone. To
get TYL concentration in the mixture, readings at 240

Fig. 8 Scheme for approach 2

nm, shown in Fig. 7, were replaced in its corresponding
regression equation.
Approach 2: Figure 8 illustrates the whole approach.
The DDRD method was investigated as another approachand found to be successfully utilized to obtain TRI. The
ternary mixture was divided using an equimolar double divisor mixture (1 µg/mL TYL + 1 µg/mL SLD), andthen derivatized. Figure 9 demonstrates this process,where readings at the coincidence point of 236.5 nm wereinserted in the relevant regression equation to obtain TRI
concentration.
Trials were conducted to obtain TYL or SLD using
DDRD but with no success, so FDRM-SS was used
to obtain the other two drugs under investigation.
FDRRM-SS was furtherly applied by obtaining SLD thensubtracting the obtained spectrum from ternary mixture
to obtain TYL.

Abdullatif et al. BMC Chemistry(2024) 18:118

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Fig. 9 The first derivative of ratio spectra of (a1) ternary mixture of SLD, TRI, and TYL,2 µg/mL each, using double divisor mixture (1 µg/mL
TYL + 1 µg/mL SLD), (a2) DD1 of 2 µg/mL pure TRI using the same double divisor, showing coincidence point at 236.5 nm

Fig. 10 First derivative spectra of both SLD and TYL using 1 µg/mL TRI as a divisor, (a) zoom in on the region from 210–240 nm (b) zoom
out to show the region from 240 to 280 nm showing SLD with ­Pmin 255.1 and TYL with ­Pmin 254.7

As shown in Fig. 10a, first derivative absorption spectrum of SLD using 1 µg/mL TRI as a divisor owned ashoulder where there was zero contribution of TYL at
225.1 nm. When the ternary mixture (SLD+TYL+TRI)
was divided by 1 µg/mL TRI and the first derivative
was obtained (TRI was cancelled), readings at 225.1 nm
were multiplied with the factorized spectrum of SLD.
The resulted spectrum represented ­DD1 SLD alone, andreadings at 255.1 nm were replaced in its corresponding regression equation. Finally, SS was applied; after

subtracting the resulted ­DD1 SLD spectrum from ­DD1 ofthe ternary mixture, the resulted spectrum would be ­DD1
TYL alone. To get TYL concentration in the mixture,readings at 254.7 nm were substituted in its corresponding regression equation, as shown in Fig. 10b.
Approach 1, where DRSZ, FDRM-SS was utilized,
showed lower manipulation steps. Approach 2 hadcertain limitations as DDRD method requires equimolar
divisor concentrations and a coincidence point where
measurements are conducted. As a conclusion, and by

Abdullatif et al. BMC Chemistry(2024) 18:118

Page 12 of 15

taking into consideration the whole process of both
approaches, the second approach was recommended as it
was more accurate and reliable.

The score demonstrates a dedication to sustainability
and furthers the better recognized goals of analyticalchemistry.

Greenness assessment

WAC evaluation model

It is obvious that nowadays, within all the uprising
environmental drastic changes and warning signs ofenvironmental fatigue, applying green chemistry [36]seems a must. The responsibility and burden of awareness,saving and looking after the environment mainly falls on
the shoulders of scientists and researchers. We believethat all researchers should ensure full compliance withthe greenness requirements and guidelines in every study
made. For instance, it’s critical to replace conventional
pharmaceutical analysis techniques with eco-friendly
approaches with comparable compliance properties,
because the former rely on extensive ingestions of manyhazardous and organic solvents. This study abided by thegreenness guidelines all through the process [37–39].
Moreover, AGREE, one of the most recent greenness
assessment instruments, was used to assess the suggested
approaches’ greenness level [40].
Analytical GREEnness metric approach and software (AGREE)

AGREE is a comprehensive and simple assessment
method which among all the greenness assessment
methods, only AGREE includes all twelve theories ofGreen Analytical Chemistry [41]. The greenness profile ofthis study was also assessed using the AGREE calculator,
and the predicted AGREE score involving all 12 different
concepts of GAC for this study is shown in Fig. 2. The
scores more than 0.75 indicate that the method is ofgreenness for the routine drug determination. Moreover,a 0.50 score implied the method is fit for routine drug
determination. Finally, scores under 0.50 indicated theunacceptableness of the proposed method. The AGREE
score for this study’s proposed electrodes of GAC was
0.93. These principles encompass reducing or eliminatinghazardous compounds, minimizing waste generation,
optimizing energy usage, and enhancing safety as wellas health. It has a great commitment to environmental
responsibility along with sustainability.
The best scores were owned by the sample treatment
and energy consumption as they were within greenness
limitations, along with all of sample preparation,
derivatization, waste, and toxicity as there was none ofthem. A medium score was given to point 12, referring to
toxicity, as methanol is highly flammable. The least scorewas given to point 2, referring to the sample amountbecause, according to the second principle of GAC, aminimal sample size and a minimal number of samples
are goals. Also point 5 refers to automation, as themethod is semi-automized, miniaturized.

White Analytical Chemistry can be conceptualized asa green approaches’ expansion, with an advantage ofevaluating whether the proposed method is “fit-forpurpose”, a logical goal all studies should strive for [42].
The approach additionally includes questions about theeconomical aspect as well as sustainable development(SD), so in other words, it reinforces greenness to go
hand in hand with economical value and sustainability.
This new approach is represented by a Red-Blue-Green(RGB) model, in which the 12 new principals are divided
among those three colors. “Green” area is related to thegreenness, under which four parameters are examined.
Then there is the “Red” area, which is related to analytical
performances, also grouped under it four parameters.
Then finally the area “Blue”, where there are fourparameters grouped related to practical benefits.
This study attributed convenient scores for each of
the 12 parameters according to the considerations of
the authors of WAC. The scores are out of 100, given toa method of zero waste or energy consumption (Greenarea), a perfect hand-held instrument or easiness ofuse (Blue area), and convenient analytical performance(Red area). Consecutively, there is an algorithm modelsumming up all the scores, giving a final average numberof (whiteness), quantifying how much the method fits the
WAC principles.
The total scores of both AGREE and WAC were verysatisfactory, revealing that the proposed methods are of
excellent greenness and that we could safely propose ourmethods to be applied in the future, without causing any
harm or damage to the environment.

Conclusions
Three methods were conducted to resolve severely
overlapped ternary mixtures based on derivative ratio
spectra. The conventional double divisor derivative
ratio (DDRD), derivative ratio zero crossing point(DRSZ), and the recent factorized derivative ratio
method (FDRM). Combination between these methods
with spectrum subtraction (SS) could overcome thechallenging overlapping spectra of the three drugs.
This work represents a green, accurate, time-saving,and successful study used to resolve the severe overlap
between three components in veterinary formulation,using various manipulating methods, while maintaining
high reproducibility and accuracy.

Abdullatif et al. BMC Chemistry(2024) 18:118

The method has a benefit over the reported method[22] in that, it was timesaving, adhered to green
principles, and didn’t demand sophisticated experimental
setup, intricate processing, or heavy reliance on organic
solvents—all of which are commonly accompanying
HPLC analytical procedures. Better recoveries of thedosage form were also found utilizing this studied
method.
This work was evaluated for the analysis of the abovementioned components in various veterinary formulations
and mixtures and is therefore considered as an alternative
tool for routine testing in quality control laboratories.

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s13065-​024-​01220-4.
Supplementary Material 1.
Supplementary Material 2.
Acknowledgements
Ahmed I. Sheta: for his generous help during brainstorming and revision ofthis work.
Author contributions
H. Abdullatif: conception, design of the work; acquisition, analysis,
interpretation of data, approved the submitted version, and agreed both tobe personally accountable for the author’s own contributions and to ensurethat questions related to the accuracy or integrity of any part of the work,
even ones in which the author was not personally involved, are appropriately
investigated, resolved, and the resolution documented in the literature. M.
Abdelkawy: revised work, approved the submitted version, and agreed bothto be personally accountable for the author’s own contributions and to ensurethat questions related to the accuracy or integrity of any part of the work,
even ones in which the author was not personally involved, are appropriately
investigated, resolved, and the resolution documented in the literature. M.
Kamal: revised work, approved the submitted version, and agreed both tobe personally accountable for the author’s own contributions and to ensurethat questions related to the accuracy or integrity of any part of the work,
even ones in which the author was not personally involved, are appropriately
investigated, resolved, and the resolution documented in the literature. N.
Fahmy: conception, design of the work, interpretation of data, approved
the submitted version, and agreed both to be personally accountable forthe author’s own contributions and to ensure that questions related to theaccuracy or integrity of any part of the work, even ones in which the author
was not personally involved, are appropriately investigated, resolved, and theresolution documented in the literature.
Funding
Open access funding provided by The Science, Technology & Innovation
Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank
(EKB). Not applicable.
Data availability
Data is provided within the manuscript or supplementary information files,orupon request, E-mail : hanoood7@hotmail.com.

Declarations
Ethics approval and consent to participate
All authors agree, and there is no participants or patients in this study “notapplicable”.

Page 13 of 15

Consent for publication
All authors agree for publication, and there is no participants or patients in thisstudy “not applicable.”
Competing interests
The authors declare no competing interests.
Received: 16 February 2024 Accepted: 3 June 2024

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