(2024) 18:129
Alnemari et al. BMC Chemistry
https://doi.org/10.1186/s13065-024-01226-y

BMC Chemistry

RESEARCH

Open Access

Application of signal processing techniques
for the spectroscopic analysis of dolutegravir
and lamivudine: a comparative assessment
and greenness appraisal
Reem M. Alnemari1, Ahmed H. Abdelazim2, Atiah H. Almalki3,4, Arwa S. Alqahtani5, Saleh I. Alaqel6,
Fahad T. Alsulami7 and Ahmed Serag2*

Abstract
HIV treatment has greatly improved over the years, with the introduction of antiretroviral drugs that target the virus
and suppress its replication. Dolutegravir and lamivudine are two such antiretroviral drugs that are commonly used
in HIV treatment regimens. Herein, three spectrophotometric methods manipulating ratio spectra were developed
for the simultaneous analysis of dolutegravir and lamivudine in their binary mixtures. These methods include
mathematical processing stages like ratio difference method or signal processing approaches such as the first
derivative of the ratio spectra, and continuous wavelet transform. The developed spectrophotometric methods
exploit the characteristic spectral differences between dolutegravir and lamivudine in order to quantify themsimultaneously. These methods have shown promising results in terms of sensitivity and selectivity as validated
per the ICH guidelines. Moreover, these methods offer a straightforward and economical alternative to more intricate
analytical methodologies like high-performance liquid chromatography. By incorporating the analytical ecoscale and AGREE for greenness evaluation of the proposed methods, we can further ensure that these techniques
are effective and environmentally friendly, aligning with the principles of green chemistry. This evaluation will
provide a comprehensive understanding of the environmental friendliness of these spectrophotometric methods
in pharmaceutical analysis.
Keywords HIV, Dolutegravir, Lamivudine, Spectroscopy, Greenness evaluation*Correspondence:
Ahmed Serag
Ahmedserag777@hotmail.com; Ahmedserag777@azhar.edu.eg
1
Department of Pharmaceutics and Industrial Pharmacy, Collegeof Pharmacy, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia
2
Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy,
Al-Azhar University, Cairo 11751, Egypt
3
Department of Pharmaceutical Chemistry, College of Pharmacy, Taif
University, P.O. Box 11099, Taif 21944, Saudi Arabia
4
Addiction and Neuroscience Research Unit, Health Science Campus, Taif
University, P.O. Box 11099, Taif 21944, Saudi Arabia
5
Department of Chemistry, College of Science, Imam Mohammad Ibn
Saud Islamic University (IMSIU), P.O. Box, 90950, Riyadh 11623, Saudi
Arabia6
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Northern Border University, Rafha 91911, Saudi Arabia
7
Department of Clinical Pharmacy, College of Pharmacy, Taif University,
P.O. Box 11099, Taif 21944, Saudi Arabia© 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
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Alnemari et al. BMC Chemistry(2024) 18:129

Introduction
The use of combination antiretroviral medication has
dramatically impacted treatment techniques for those
infected with the human immunodeficiency virus (HIV)[1]. There are now around twenty-five antiretroviral
medicines classified into six broad groups: proteaseinhibitors, integrase strand transfer inhibitors, CCR5
antagonists, fusion inhibitors, nucleotide/nucleosidereverse transcriptase inhibitors (NTRI), and nonnucleoside reverse transcriptase inhibitors. Combining
nucleotide/nucleoside reverse transcriptase inhibitorswith additional drugs is a popular method for HIV
therapy [2]. Lamivudine (LMV), an example of an NTRI,
has demonstrated success in treating HIV infections
and is frequently selected as the initial or subsequent
medication choice due to its tolerance and long-term
safety record [3]. LMV has recently been utilized totreat HIV patients in conjunction with the integrase
inhibitor dolutegravir (DTG) [4]. Clinical investigations
have shown that this combination successfully reduces
viral loads in those who are infected at high levels.
Furthermore, virologic suppression has been achieved
and sustained in diverse HIV populations, including
those moving from a three-drug regimen, using a twodrug regimen strategy consisting of DTG+LMV [5].
Limited research has been conducted on theanalytical methodologies for determining the
combination of DTG and LMV in their combined
dosage forms. Previous studies have primarily utilized
chromatographic separation techniques, including
high-performance liquid chromatography (HPLC) andliquid chromatography-tandem mass spectrometry(LC–MS/MS), to analyze these drugs [6–8]. However,
it is important to note that these chromatographic
methods do possess limitations such as environmentally
unfriendly practices due to the significant use of organic
solvents and time-consuming optimization processes.
UV spectroscopy, on the other hand, offers various
advantages, including simplicity, fast analysis durations,and the demand for minimal solvent quantities, making
it a more environmentally friendly alternative tochromatographic procedures [9–11]. Furthermore, bymerging mathematical manipulation processing stages
with spectroscopic analysis, spectrum overlapping
concerns may be addressed. The majority of theseprocedures rely on mathematical formulae or signal
processing of the spectra, with no need for chemicalor physicochemical separation [12–15]. However, very
little literature has reported the application of suchmethods in DTG and LMV quantitative analysis [16–18]. One of the main drawbacks of these methods istheir greenness evaluation as methanol was employed
as the main solvent which is known to be hazardous

Page 2 of 13

and toxic. Also, none of these methods evaluated somekey validation parameters such as the selectivity andspecificity of the developed procedures. Besides, somespecialized software and experimental designs must be
implemented particularly for the chemometric-assisted
methods for DTG and LMV analysis.
As a result, the purpose of the current study is todevelop sensitive and selective spectrophotometricmethods for the analysis of these medications basedon mathematical and signal processing procedures
manipulating ratio spectra, such as ratio difference (RD),first derivative of the ratio spectra (1DD), and continuous
wavelet transform (CWT) posing them as powerfulalternatives to the reported methods especially inresources limited settings. The suggested techniques will
also be evaluated for their greenness using two metrics
systems, the analytical eco-scale [19] and greenness
metric (AGREE) [20], to ensure their environmental
friendliness.

Experimental
Materials and solvents

Dolutegravir and lamivudine were provided by the
Egyptian Drug Authority (EDA) with purities of 98.7 ± 0.5
and 99.4 ± 0.5, respectively. The dosage form used foranalysis was D­ ovato® tablets (50 mg DTG and 300 mg
LMV), manufactured by ViiV Healthcare LLC in the UK.
HPLC-grade ethanol was sourced from Sigma-Aldrich inDarmstadt, Germany.
Instrumentation and software

A Shimadzu UV–Visible 1800 Spectrophotometer was
employed for the measurements, with the samples’
absorption spectra being scanned using 10 mm quartz
cuvettes. The spectral scan speed was set to fast withbandwidth of 1 nm, and distance between points i.e. datainterval of 0.2 nm subsequently, the scanned spectra
were obtained in the range of 200–400 nm and analyzed
using Shimadzu UV-Probe software version 2.43. The
CWT method was implemented in MATLAB R2015a(8.5.0.197613) employing the wavelet toolbox.
Standard solutions

In order to prepare standard stock solutions for DTG
and LMV, 10 mg of each drug powder was separately
dissolved in the minimal quantity of ethanol. The
resultant solutions were subsequently diluted to a final
volume of 100 mL with distilled water using separate
100-mL volumetric flasks. Consequently, both solutions
had concentrations of 100 μg/mL.

Alnemari et al. BMC Chemistry(2024) 18:129

Procedures
Linearity and calibration graphs

To determine the concentration of DTG and LMV,
precise volumes from their working standard solutions
were transferred into separate 10-mL volumetric flasks
to prepare a series of standard solutions of each drug inthe concentration ranges of 1.5–32 and 1.5–36 μg/mL for
DTG and LMV, respectively. Distilled water was added
to each flask to reach the desired volume. Subsequently,
the absorption spectra of these diluted solutions were
measured in the wavelength range of 200–400 nm using
distilled water as a reference blank. To obtain the ratio
spectra of DTG and LMV, the absorption spectra of eachdrug were divided by a suitable divisor spectrum fromthe other drug. For DTG, a spectrum of 12 µg/mL LMV
was found to be optimal, while for LMV, a spectrum of
18 µg/mL DTG was used.
For the ratio difference method (RD): The differencesin amplitudes between specific wavelength pairs (258
& 229 nm for DTG and 281 & 241 nm for LMV) in theratio spectra of each drug were plotted against theircorresponding concentrations to create calibrationgraphs. Linear regression equations were derived fromthese plots to estimate the concentrations of DTG and
LMV accurately.
For the first derivative of the ratio spectra method (1DD):
The obtained ratio spectra of each drug were converted totheir first-order derivative using a wavelength difference(∆λ) of 4 and a scaling factor of 100. The amplitudes at
271 nm for DTG and at 289 nm for LMV were recorded
from the derived derivative spectra. These recorded
values were plotted against different concentrations ofeach drug to construct calibration graphs, from which
regression equations were obtained.
For the continuous wavelet transform method (CWT):The ratio spectra obtained for each drug were converted
to the wavelet domain using the bior 2.4 wavelet family
and a scaling factor of 20 under Matlab environment.
The amplitudes at 259 nm for DTG and at 281 nm for
LMV were recorded from the transformed spectra.
The obtained values were plotted against various
concentrations of each drug to create calibration graphs,
which resulted in the derivation of regression equations.
Validation of the developed methods

Validation of the developed methods was conductedfollowing ICH Q2 (R1) guidelines for validation ofanalytical procedures [21]. Linearity was assessed using
nine different DTG and LMV concentrations with theprocedures mentioned in Sect. "Linearity and calibrationgraphs". The resulting calibration curve yielded values
for correlation coefficient (R2), slope, and y-intercept.

Page 3 of 13

Limits of detection and quantification were determinedbased on standard deviation of the response, calculated
from residuals, and slope of the calibration curve using
LOD = 3 × (σ/S) and LOQ = 10 × (σ/S), where σ is thestandard deviation of the response and S is the slope.
The accuracy and precision were evaluated bycomparing measured concentrations with known ones
for three replicates at levels of 6, 12, and 18 µg/mL
to determine percentage recovery as well as relative
standard deviation (% RSD). To assess the specificity ofthe developed techniques, different concentrations of
DTG and LMV from reference solutions were transferred
into separate 10-mL volumetric flasks and diluted withwater. The samples were then subjected to analysis using
the prescribed protocols for linearity and calibration
curves for each drug, enabling computation of theirindividual concentrations.
Greenness evaluation of the developed methods

The greenness evaluation of the proposed methods
was conducted based on established criteria forenvironmentallyfriendly
analyticaltechniques.
Analytical Eco-Scale was used to assess the greennessof the proposed methods based on criteria such aswaste generation, energy consumption, and the use ofhazardous materials [19]. AGREE prep software wasalso utilized to evaluate the environmental impact of the
proposed methods by considering the 12 principles ofgreen chemistry [20].
Analysis of pharmaceutical tablets

To determine the concentration of DTG and LMV,
five ­Dovato® tablets weighing 50 mg DTG and 300 mg
LMV each were pulverized into a fine powder. A precise
quantity equivalent to 5 mg DTG and 30 mg LMV wasthen transferred to a 100-mL volumetric flask containing
40 mL of ethanol. After vigorous shaking for 20 min,the mixture was filtered. Additional ethanol was used
to adjust the final solution volume as per desiredconcentration, followed by subsequent dilutions withwater resulting in the preparation of five samples withvarying concentrations. Each sample underwent analysis
following specified procedures as described in linearity
assessment and calibration curve plotting method
protocols, allowing computational determination of drugconcentrations present in the samples.

Results and discussion
Spectral characteristic

The UV spectrum of DTG exhibits two significant
absorption peaks in the UV region: one at 260 nm andanother at 336 nm. These peaks can be linked to theelectronic structure of DTG (Fig. 1). Specifically, theAlnemari et al. BMC Chemistry(2024) 18:129

Page 4 of 13

Fig. 1 Zero order absorption spectra of 12 μg/mL DTG & 12 μg/mL LMV showing severe overlapping

peak at 260 nm is likely due to the electronic transitionsin the aromatic rings and the other pi-conjugatedsystems within the compound. Meanwhile, the peak at
340 nm could be attributed to n-pi* transitions, which
involve the non-bonding electrons of electronegative
atoms of DTG such as oxygen and nitrogen. These
functional groups absorb at slightly longer wavelengths
than the pi-pi* transitions of aromatic rings. On the
Other hand, LMV shows only a single absorption peakin the UV region at around 270 nm (Fig. 1), which
can be attributed to the pi-pi* electronic transitionsof the cytosine ring—a heterocyclic aromatic ringwith both carbon and nitrogen—it’s this part of LMV
that is likely responsible for the absorption at 270 nm.
As a consequence of these results, DTG and LMV
exhibit significant overlap in their absorption spectra
at zero-order, making it challenging to accurately
determine the concentration of each drug (Fig. 1).
In order to overcome this issue, three mathematical
spectrophotometric methods have been developed tomanipulate their ratio spectra by applying constantremoval of the interfering compound i.e. the otherdrug either via mathematical processing techniques
like RD method or signal processing approaches such
as 1DD and CWT. Implementing these preprocessing
steps assists in eliminating interfering components
without prior separation, thus facilitating the accurate

measurement of DTG and LMV in their combined
dosage form.
Methods development and optimization

The RD method relies on the use of a mixture
containing two drugs, X and Y, with overlapping
spectra. To determine the presence of X, the spectrumof the mixture is divided by a known concentration of
Y. This division produces a new curve representing X/Y’and Y/Y’, where Y/Y’ remains constant. By choosing two
wavelengths λ1 and λ2 from this ratio spectrum andsubtracting their amplitudes, any constant values alongwith instrumental errors or sample matrix interferences
can be eliminated [22]. Hence, this method is
influenced by two main factors: the concentrationof the divisor and the selection of wavelengths. The
concentration of the divisor plays a crucial role indetermining the structure of the obtained ratio spectra
for DTG and LMV. After careful examination, it wasdetermined that divisors concentrations of 12 μg/
mL for LMV and 18 μg/mL for DTG exhibited higher
linearity with minimal noise and improved sensitivity.
Additionally, choosing appropriate wavelength pairs
is vital as it affects important analytical parameterssuch as intercept, slope, and correlation coefficient in
the derived calibration curves. Different wavelength
pairs were tested for each drug, revealing that using

Alnemari et al. BMC Chemistry(2024) 18:129

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Fig. 2 Ratio spectra of DTG in the range of 1.5–32 µg/mL using 12 µg/mL LMV as a divisor

Fig. 3 Ratio spectra of LMV in the range of 1.5–36 µg/mL using 18 µg/mL DTG as a divisor(258 & 229 nm) for DTG and (281 & 241 nm) for LMV
resulted in optimal outcomes, as depicted in Figs. 2
and 3. Then, the regression equations were derived
by constructing calibration curves that plotted thedifference in amplitude at these specific wavelengths
against the corresponding concentrations of DTG and
LMV, respectively (Table 1).

Another method that can be utilized to determine
the concentration of DTG and LMV is derivative
spectrophotometry, which functions as a signal
processing technique. Derivative spectrophotometry
is an analytical approach that proves advantageous inresolving spectral overlap by analyzing higher derivativesof absorbance with respect to wavelength. By applying

Alnemari et al. BMC Chemistry(2024) 18:129

Page 6 of 13

Table 1 Regression and validation data for quantitative analysis of DTG and LMV by the proposed methods
Parameters

1

RD
DTG

LMV

DD

DTG

CWT​
LMV

DTG

LMV

Wavelength (nm)229 & 258

240 & 281

271

289

259

281

Linearity range (µg/mL)

1.5–32

1.5–36

1.5–32

1.5–36

1.5–32

1.5–36Slope

0.02160.08050.5064

1.1566

0.4076

1.418

Intercept

0.00070.00630.02350.08770.01420.1074

Coefficient of determination ­(r2)

0.99990.99990.99990.99990.99990.9999LOD (µg/mL)

0.330

0.477

0.330

0.478

0.358

0.473

LOQ (µg/mL)

1.000

1.445

0.999

1.449

1.085

1.433

Accuracy (%R)a99.61

100.35

98.83

99.99

100.83

99.99

Repeatability precision (%RSD)b

0.411

0.347

0.299

0.318

0.693

0.414

Intermediate precision (%RSD)b

0.589

0.401

0.351

0.423

0.862

0.736

a

Average of 9 determinations (3 concentrations repeated 3 times)b

RSD of 9 determinations (3 concentrations repeated 3 times)

Fig. 4 1D ratio spectra of DTG in the range of 1.5–32 µg/mL using 12 µg/mL LMV as a divisor

the first derivative to the ratio spectra data of both drugs,
it becomes possible to quantify DTG at 271 nm and LMV
at 289 nm, as depicted in Figs. 4 and 5. To investigate theinfluence of instrumental parameters on the formation ofderivative spectra, we conducted experiments to explore
different smoothing and scaling factors. Our findings
revealed that using a Δλ value of 4 nm and a scaling
factor of 100 produced optimal results. Subsequently,
regression equations were computed based on thefirst derivative of ratio spectra (1DD) for each drug inrelation to their final concentrations. These equations are
summarized in Table 1.
Another signal processing approach is the CWT
which was employed for analyzing DTG and LMV in
their binary mixture. The wavelet transform represents
a powerful mathematical approach used in signal
processing and data analysis. Its primary aim is toremove any constant elements or interference from thesample matrix, enabling accurate determination of drug
X concentration within a mixture. The fundamental

Alnemari et al. BMC Chemistry(2024) 18:129

Page 7 of 13

Fig. 5 1D ratio spectra of LMV in the range of 1.5–36 µg/mL using 18 µg/mL DTG as a divisor

concept involves expressing any arbitrary function as
a combination of wavelets, defined as functions Ψa,b(λ) derived from a base function Ψ(λ) through dilationand shift [23]. This base function is often referred toas the “mother” wavelet since it generates a family of
wavelets. Various families of wavelet bases exist, suchas Daubecies, Symlet, Coiflet, Meyer etc., with themother wavelet generating functions Ψa,b (λ) by scaling
and shifting. The CWT produces multiple coefficients
C that are dependent on scale (s) and position (τ).
Multiplying each coefficient by appropriately scaled andshifted wavelets yields constituent components from theoriginal signal—a process applicable for decomposing
drug mixture spectra to identify specific contributions
from each drug. In this context, various wavelet families
such as Coiflet (Coif), Biorthogonal (bior), Daubechies(Db), Gaussian, etc., were tested on the ratio spectra atdifferent orders and with different scaling parameters.
The goal was to obtain acceptable calibration curves andensure accurate determination of DTG and LMV. After
thorough testing, it was determined that the bior wavelet
family of second order (2.2) with a scaling factor (a) of
20 yielded the best results for DTG and LMV analyses.
Subsequently, CWT spectra were generated by plotting
the obtained CWT-coefficients against wavelengths asshown in Figs. 6 and 7. To construct calibration graphs
for DTG and LMV, transformed signals’ amplitudes
were plotted against their corresponding concentrations

at 256 nm and 218 nm, respectively which led tothe derivation of the regression equations for bothcompounds (Table 1).
Methods validation

Validation of the developed spectrophotometric methods
was conducted to assess their accuracy, precision, andselectivity following the ICH guidelines [21]. In thiscontext, the linearity of the calibration curves wasevaluated by analyzing a series of standard solutions
with known concentrations of DTG and LMV. The
results showed that all the developed methods exhibited
good linearity over the concentration range 1.5–32 µg/
mL and 1.5–36 µg/mL for DTG and LMV, respectively.
Additionally, the methods showed high sensitivity
exemplified by their low detection limits for DTG
ranging from 0.330 to 0.358 µg/mL and for LMV ranging
from 0.473 to 0.478 µg/mL, respectively (Table 1).
Regarding accuracy, both DTG and LMV showed percent
recovery values ranging from 98 to 102%, suggestingthat the established methods offer excellent results inaccurately determining their concentrations. Moreover,the precision of the developed spectrophotometricmethods was evaluated by calculating the percent relative
standard deviation (% RSD) of replicate measurements
for each drug over several different concentrations. The
percentage relative standard deviation (% RSD) values
for these methods were found to be within acceptable

Alnemari et al. BMC Chemistry(2024) 18:129

Fig. 6 Continuous wavelet transform ratio spectra of DTG in the range of 1.5–32 µg/mL using 12 µg/mL LMV as a divisor

Fig. 7 Continuous wavelet transform ratio spectra of LMV in the range of 1.5–36 µg/mL using 18 µg/mL DTG as a divisor

Page 8 of 13

Alnemari et al. BMC Chemistry(2024) 18:129

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LC–MS/MS (22 penalty points), HPLC (19 penalty
points), UPLC (18 penalty points) and even the otherreported spectrophotometric methods (9 penalty points),suggesting outstanding green analysis of the developed
methods (Table 3). Such results could be attributed tothe careful consideration of green chemistry principles
in the selection of reagents and instrumentation of
the developed spectrophotometric methods, leading
to reduced environmental impact. On the other hand,most of the reported chromatographic techniquessuch as LC–MS/MS, HPLC, and UPLC were found tohave higher penalty point scores due to excessive use ofhazardous reagents such as methanol and acetonitrile
in addition to higher energy consumption. The reported
spectrophotometric methods also had higher penalty
point scores compared to the developed methods,
indicating they are less environmentally friendly due tothe employment of methanol as a solvent.
The other metric system: AGREE is an easily accessible
software tool that adheres to the principles of green
analytical chemistry and produces a clock-like graphoutput based on twelve input criteria, displaying
an overall score and color representation, where aperfect analysis would attain a score of one and be
depicted by a deep green color. The results obtained
from AGREE displayed in Table 3 indicate that the
proposed spectrophotometric methods achieved an
impressive score of 0.79, while the chromatographic
methods mentioned in literature received a yellow
representation with a comparatively lower scores of
0.49, 0.57 and 0.55 for the LC–MS/MS, UPLC andHPLC methods, respectively. This finding implies that

limits, indicating good precision (Table 1). In addition
to linearity, accuracy and precision, the selectivity of the
proposed methods was examined by analyzing different
laboratory-prepared mixtures with varying ratios of DTG
and LMV using the suggested techniques. The findingspresented in Table 2 indicate that both drugs can be
accurately measured without any interference from eachother, thereby confirming satisfactory selectivity.
Greenness assessment of the developed methods

The environmental compatibility of the proposed
methods in comparison with the reported methods
was also assessed using green chemistry principles via
implementing two metric approaches, namely analyticaleco-scale and AGREE metrics [24]. The analyticaleco-scale metric calculates penalty points based on
two primary analytical parameters. One parameter isrelated to the reagents used, which can be estimated byconsidering their quantities as well as environmental
hazards. The other parameter is associated with the
instrumentation used, that depends on the energy
consumption during operation, and waste generated by
the device. After calculating these penalty point scores,
the results are subtracted from 100 to determine whether
the approach qualifies as outstanding (green), reasonable(semi-green), or inadequate in terms of its environmental
sustainability. Using this greenness assessment tool,
the developed analytical methods for the quantitative
measurement of DTG and LMV in their pharmaceuticalpreparation were evaluated. The results indicate that thedeveloped methods yielded a score of 3 penalty points
in comparison to the reported methods including theTable 2 Application of the proposed methods for determining DTG and LMV in their laboratory mixed solutions
Added (µg/mL)%Recovery
1

RD
DTG

LMV

DTG

DD

LMV

DTG

CWT​
LMV

DTG

LMV

2

12

100.23100.07

98.59

99.99

98.71

98.44

3

18

99.23

101.01

98.57

101.0099.23

100.52

4

24

98.73

100.2498.02

100.21

98.61

98.41

6

18

99.00

100.32

98.62

100.29

98.85

99.33

8

24

99.71

100.6699.02

100.61

99.76

100.586

6100.59

99.76

98.82

99.41

99.37

99.27

6

12

101.48100.89100.11

99.75

101.15100.08

8

16

98.24

98.09

101.93100.9898.18

100.9812

6100.23100.91

98.93

100.40

98.18

99.63

18

6

101.57

99.22

99.96

99.32

99.18

100.69

24

8

98.9

100.22101.6599.69

101.29100.45

24

4

98.74

100.52

99.71

99.84

100.24100.6899.72 ± 1.102

100.16 ± 0.830

99.49 ± 1.237

100.12 ± 0.558

99.40 ± 1.041

99.92 ± 0.890

Mean ± SD

Alnemari et al. BMC Chemistry(2024) 18:129

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Table 3 Greenness evaluation and comparison of the proposed method and reported methods using the described metrics
MethodAnalytical eco-scale
Parameters

Proposed method

AGREE tool
Penalty points

Reagents
Water

0

Instrument spectrophotometry
  Energy consumption0  Occupational hazards

0  Waste3∑3

Total scores

100–3 = 97

Spectrophotometric methods 16–18 Reagents
Methanol6

Phosphate buffer pH 6.8

0

Water

0

Instrument spectrophotometry
  Energy consumption0  Occupational hazards

0  Waste3∑9

Total scores
LC–MS/MS 7

100–9 = 91

Reagents
Acetonitrile

8

Formic acid6

Instrument LC–MS/MS  Energy consumption2

  Occupational hazards

0  Waste6∑22

  Total scores
UPLC 8100–22 = 78

Reagents
Methanol

12

 ­K+ dihydrogen orthophosphate0Instrument UPLC
  Energy consumption0  Occupational hazards

0  Waste6∑18

  Total scores
HPLC 6100–18 = 82

Reagents
Methanol

12

Sodium dihydrogen phosphate0Instrument HPLC
  Energy consumption1  Occupational hazards

0  Waste6∑19

  Total scores

100–19 = 81

Alnemari et al. BMC Chemistry(2024) 18:129

Page 11 of 13

compared to existing chromatographic methods, ourproposed methods exhibit greener characteristics. The
greenness evaluation of our proposed methods has also
been compared with the reported spectrophotometricmethods. The results showed that our proposed
methods exhibited higher greenness scores comparedto the reported spectrophotometric methods, indicating
their superior eco-friendly characteristics. The major
limitations in the greenness profile of the developed
methods are related to items 1, 3, 7 and 10 of the AGREE
analysis. Items 1 and 3 are related to the sampling siteand position of the spectrophotometer; they require
careful consideration as they should be performed offsite or in a controlled environment. This is why they wererepresented in yellow and orange colors, respectively.
Furthermore, items 7 and 10 colored in yellow areassociated with the amount of waste and types of reagentsused. Although some of our reagents are bio-based suchas water, they may still contribute to waste generation;
therefore, proper management and disposal is necessary.
In conclusion, the proposed spectrophotometric methods
demonstrate superior eco-friendly characteristics
compared to existing chromatographic methods
and reported spectrophotometric methods based on
greenness evaluation using the described metrics.
Application to pharmaceutical formulation and greenness
assessment

The developed methods utilized in this study effectively
measured the concentrations of DTG and LMV in theirrespective pharmaceutical dosage forms. The obtained
results were compared to those from a previously
reported chromatographic method, revealing a highlevel of agreement (Table 4). To further assess any
potential interference caused by excipients, the standard
addition technique was employed, ultimately yielding
excellent recoveries for both DTG and LMV (Table 4).

Additionally, a comprehensive statistical analysis was
conducted using t-tests and F-tests to compare thedeveloped spectrophotometric methods with thereported chromatographic method. The results indicatedthat there is no significant difference between the two
approaches, as evidenced by lower-than-expected
t-values and F-values. A summary of these findings canbe found in Table 4.

Conclusion and future directions
In conclusion, this research article presents thedevelopment and evaluation of three spectrophotometricmethods for the simultaneous analysis of DTG and LMVin their binary mixtures. The methods, including RD,
1
DD, and CWT, demonstrated significant potential interms of simplicity, sensitivity, selectivity, and greenness
profiles in quantifying the two antiretroviral drugs
compared to reported spectrophotometric methods.
In detail, the RD method offers a significant advantage
as it can be applied at any two wavelengths throughout
the entire ratio spectrum, without being affected byoverlapped components. Moreover, the use of 1DD
corrects for interferences and ultimately enhances
the purity of analytical peaks. This is particularly
advantageous when dealing with high levels of interfering
compounds. On the other hand, the CWT improves
signal-to-noise ratio and has multiple families available
that cater to a wide range of applications, making ita highly promising approach for manipulating ratio
spectra. Additionally, the developed methods offer asimpler and more cost-effective alternative to highperformance liquid chromatography. It is worth
mentioning that the 1DD transcended the other methods
in terms of accuracy, precision and sensitivity posing
this method as a potential candidate for routine analysis
of DTG and LMV in pharmaceutical formulations. The
incorporation of the analytical eco-scale and AGREE

Table 4 Quantitative analysis of DTG/LMV in commercial ­Dovato® tablets by the proposed methods and statistical comparison withthe reported methods
DTG/LMV%Recovery ± RSD
1

RDDovato® ­tabletsa

DD

Reported method 6

CWT​

DTG

LMV

DTG

LMV

DTG

LMV

DTG

LMV

99.67 ± 0.823

99.55 ± 0.849

99.36 ± 1.037

99.91 ± 0.729

99.11 ± 0.552

99.57 ± 0.700

99.21 ± 0.919

99.78 ± 0.659

Standard ­additionb

99.24 ± 0.609

100.99 ± 0.676

98.42 ± 0.311

100.94 ± 0.672

99.55 ± 0.536

100.28 ± 1.202

t-test (2.101)c

0.756

1.029

1.809

0.067

1.192

0.692

F-test (3.179)c

1.576

1.343

2.251

1.471

1.233

1.650

a

Average of ten determinationsb

Average of three determinationsc

The values in parenthesis are tabulated values of “t “and “F” at (P = 0.05)

Alnemari et al. BMC Chemistry(2024) 18:129

for greenness evaluation further ensures that these
spectrophotometric methods are more environmentallyfriendly than the chromatographic procedures. These
results contribute to the field of pharmaceuticalanalysis by providing a comprehensive understandingof the greenness and practicality of spectrophotometricmethods for the analysis of these two antiretroviraltherapies.
Future studies could explore the application ofthese methods to analyze other antiretroviral drugs
or different combinations of drugs. Additionally,
it would be beneficial to investigate other related
UV spectrophotometric techniques, such as diffuse
reflectance spectroscopy, for the analysis of DTG and
LMV in their solid forms. This technique has shown
potential for direct analysis of solid samples [25], which
could provide a more convenient and rapid method for
quality control purposes.
Acknowledgements
The authors extend their appreciation to the Deanship of Scientific Research
at Northern Border University, Arar, KSA for funding this research work throughthe project number “NBU-FFR-2023-0175”.
Author contributions
A.S. and A.H.Abdelazim. provided study concept and design. A.H.Abdelazim
and R.M.A. performed the experiments, R.M.A. and A.H.Almalki. analyzed thedata. A.S.A. and S.I.A. interpreted the data. A.S., R.M.A. and F.T.A. wrote themanuscript. A.H.Almalki and S.I.A. revised the manuscript and data. All authors
read and approved the final manuscript.

Page 12 of 13

6.

7.

8.

9.

10.

11.

12.

13.

Funding
Deanship of Scientific Research at Northern Border University, Arar, KSA
through the project number “NBU-FFR-2023-0175”.

14.

Data availability
The data presented in this study are available on request from thecorresponding author.

15.

Declarations

16.

Competing interests
The authors declare no conflict of interest.

17.

Received: 6 February 2024 Accepted: 13 June 2024

18.

19.
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