Derivatizing agents for spectrophotometric and spectrofluorimetric determination of pharmaceuticals: a review

Most drug structures lack appropriate chromophores that can be examined at wavelengths other than the generic UV part of the electromagnetic spectrum. Indirect spectrophotometric/spectrofluorimetric analysis involving chemical derivatization in which molecules lacking UV-Vis absorbing moieties are transformed into derivatives with good chromophores. The article highlights the discussion of significant derivatizing agents for the derivatization of drug molecules that lack the chromophoric moiety required for spectrometric analysis. In addition, the probable reaction for colour generation is also discussed. The current developments of derivatizing agents for the spectrophotometric determination of pharmaceuticals are considered.


Introduction
Current analytical chemistry is constantly pushing the limits of ultrasensitive investigation to lower detection values.Spectrometry is considered a reliable and accurate tool in pharmaceutical investigations, particularly in simple matrixes and routine quality control analysis due to its simplicity, low expense, and short response period [1][2][3][4][5][6][7][8].The main advantage of such methods is that they enable the simultaneous examination of mixture constituents without chemical pre-treatment or visible spectrum operations like derivative and ratio spectra derivation [9][10][11].However, the structures of most drugs lack appropriate chromophores that are unable to make them examined at wavelengths other than the generic UV part of the electromagnetic spectrum [12].Therefore, derivatization procedures transform these drugs into easily established molecules with characteristics and quantities that may be connected to the parent molecules [13].Most relevant analytes are linked in their dose forms with other substances absorbed in the same spectral zone, making it impossible to measure their concentrations using conventional Ultraviolet-Visible (UV-vis) spectral studies [14].
Compounds with Low UV absorbance can be transformed into susceptible products (with λmax in/around 254 nm) through chemical derivatization.Several chemical reagents were employed to incorporate a chromophoric moiety into specific functionalities [15].The derivatizing reagents such as m-toluoyl chloride and benzoyl chloride were employed for inserting an aromatic chromophoric moiety into a coloured UV-absorbing derivative and are commonly used to identify aliphatic amines and amino acids [16].Chromophoric moieties have also been introduced to an amine group using nitrobenzoyl reagents [17].Derivatization processes are necessary to improve the identification of elusive analytes [18].The fact there are so many widely available derivatizing agents indicates that there must be a benefit to derivatization that outweighs the additional time and financial expenses involved in obtaining, managing, and preserving the reagents [19][20][21].The central tenet of derivatization is that an appropriate reaction should alter the analytical moiety's essential chemical or physical structure.Better detection and separation can be accomplished by identifying substances for a particular detection purpose or by changing a functional group to improve certain chromatographic features.The form of derivatization is greatly influenced by the analytical technique and the type of substance being studied [22].
During the last few years, a wide range of fluorescent or chromogenic agents has been frequently employed in the spectrophotometric quantitative and qualitative estimation of pharmacological medicinal substances.Functional groups contained in the drug substances are accountable for the characteristics of compounds and evaluate the recognition processes and techniques of quantification of pharmaceuticals.Understanding the methods for identifying structural features allows researchers to examine any pharmaceutical substance with a complex structure [23][24][25].
A plethora of derivatizing agents (DAs) was produced from our lab, with numerous of these being used for applications in analytical sciences, often for chromatographic separations requiring high sensitivity [26][27][28][29][30][31][32][33].The review published by Adegoke in 2012 provided an overview of derivatization reactions in spectrophotometric analysis.However, the scope of this review was limited only to the scheme of derivatization reactions with different derivatizing agents reported till 2010 [34].
For instance, biomolecules like amino acids, proteins, and nucleic acids possess reactive -NH 2 groups which can be further derivatized with the chromogenic group; thus, it becomes possible to detect these compounds using various analytical techniques [35,36].A broad range of DAs for amine derivatization has been described, most based on the conventional dye families with amine functionalities, including succinimidyl and sulfosuccinimidyl esters [37].To conquer this, the suitable alternative would be a "fluorogenic" reagent, i.e. one that emits light only after the derivatization, attempting to avoid both cross-contamination throughout spectrometric assessments and the use of comprehensive purifying practices [38].
The necessity for a considerable surplus of this thiol component, together with the blue fluorescence of products, significantly restricts the use of this technique, particularly for the chemical modification of aminobased compounds with higher noise fluorescence [39].Consequently, in complement to exhibiting low fluorescence in the emission spectrum of the derivative, critical quality reagents should be their capacity to produce a strong signal in the red or near-infrared area to prevent photoluminescence from biological systems [40].This might be accomplished by either significantly rearranging the chemical scaffolding or extending it with a p-conjugated system [41] .As a result of these observations, the scaffold's potential as a fluorescence DA for the spectrophotometric determination of aliphatic amines was investigated [42].The chemiluminescent and fluorogenic detection methods would operate on the in-situ creation of an extended pyrrole moiety, expanding the conjugation and establishing a push-pull connection between the electron-donating amine group and the electron-withdrawing -NO 2 group [43].As a result of the enforced structural modification, the absorption maximum is projected to move towards the higher wavelength of the electromagnetic spectrum, making derivatizing agents an attractive choice for amine chemical modification.
Numerous derivatizing agents have been applied for analyzing pharmaceuticals in biological specimens, and each has merits and limitations.In this article, we describe the current developments in the indirect spectrophotometric estimation of pharmaceuticals using derivatizing agents during the last few years.This is to state that no attempt has been made to review the importance and application of derivatizing agents for determining pharmaceutically significant compounds.The present article highlights the significance of various derivatizing agents for the derivatization of drug molecules that lack the chromophoric moiety required for spectrometric analysis.The probable mechanism of colour generation is also discussed.

NBD-Cl (4-chloro-7-nitro benzofurazan) as derivatizing agent
The collection of derivatizing reagents known as substituted benzooxadiazoles, which includes 4-chloro-7nitro-2,1,3-benzoxadiazole (NBD-Cl), is crucial in the field of analytical chemistry [44].The electron-withdra wing group -NO 2 can combine to produce NBD compounds of nitrobenzooxadiazole.A dimethylamino sulfonyl group (DBD) or an amino sulfonyl group (ABD) could be introduced for chemical derivatization.NBD-Cl-based derivatizing agents function by substituting Cl for the derivatization of amines or amino acids where the analyte derivative is strongly fluorescent and could be easily detected by spectrophotometry [45].To allow the identification of non-responding substances like amino acids and amino sugars by fluorescence and UV-Vis spectrometry, NBD-Cl, an activated halide, was utilized for the pre-column derivatization of certain amines, amino acids, and amino sugars [46].Acetylcysteine and captopril are two slightly absorbed substances that do not have much UV absorbance.In the borate solution, these molecules' sulfhydryl groups are used to derivatize them with (NBD-Cl).The resulting yellow derivatives have clearly defined UV-Vis absorbance curves.NBD-Cl in a methanolic solution, buffered to pH 9, appears to be a common template production method for NBD compounds [47].
The NBD-F/Cl-based reagents are also crucial beca use they can serve as precursors for producing other valuable derivatizing agents, such as the hydrazinofunctionalized benzooxadiazoles [48].The NBD (NBDH) hydrazine adducts have been used for a while.
Derivatizing compounds based on benzooxadiazole have been used for bioanalytical, environmental, and air monitoring purposes [49].Only a few benzooxadiazole compounds have good fluorescence properties, even though all benzooxadiazoles are excellent chromophores with absorption peaks in a range > 350 nm and high molar absorptivities [50].One such benzoderivative, 4-nitro-7-piperazine-2,1,3-benzo oxadiazole (NBDPZ), has been used to recognize carboxylic acids due to its excellent fluorescence characteristics.
NBD-Cl (Figure 1) is used as a derivatizing agent for estimating drugs comprising primary and secondary NH 2 groups [51][52][53].Omar and co-workers developed two spectrophotometric approaches for determining labetalol hydrochloride (LBT) in native form and dosage formulation [54].LBT, chemically, 2-hydroxy-5-[1-hydroxy-2-(1-methyl-3-phenylpropylamino) ethyl] benzamide hydrochloride (Figure 1), used as a first-line treatment for hypertension disorders like pre-eclampsia [55,56].The two approaches involve a reaction between active pharmaceutical ingredients (API) and NBD-Cl in a basic medium (pH = 7.5).LBT possesses a 2 o -NH 2 group which on reaction with NBD-Cl in a basic medium to produce a yellow-green coloured product that displayed maxima of fluorescence intensity (FI) at 540 nm after excitation at 476 nm and the absorbance maxima (e max ) of the derivative was recorded at 480 nm.
In the first method, the product obtained was analyzed through a spectrophotometer at 540 nm after excitation at 476 nm whereas in the second method measurement was carried out after excitation at 480 nm.It was observed that the FI and the e max of the derivative are significantly varied in pH.The maximum FI was observed in the pH range between 7.3 and 7.7.Whereas pH outside this range caused a decrement in both FI and e max .The derivative formed with 0.8 ml of 0.2% w/v NBD-Cl showed maximum absorption and beyond this composition, e max remains constant.For simplification 1.0 ml of 0.2%, w/v NBD-Cl was utilized for derivatization in both approaches.Validation studies indicated that the first and second method was linear between 0.1-2.0 and 1.0-11.0µg mL −1 , respectively.The suggested approaches were acceptable in evaluating commercialized medications without the influence of typical additives.Moreover, the spectrophotometric approach can be used to determine LBT in the spiked human plasma sample.
Recently, Anwer and co-workers used NBD-Cl to derivatize 6-Aminocaproic acid (ACA) for its determination in biofluids [57].ACA is a hydrophobic ω-amino acid with a flexible structural framework.The derivatization involves a substitution reaction between the 1 o amine of ACA with NBD-Cl to yield a yellow derivative.The reaction occurred in borate buffer (pH 9) and its FI was observed at 525 nm after excitation at 472 nm.The method was reported to be linear in the concentration range of 0.1-0.7 µg mL −1 with LOQ and LOD down to 0.101 µg mL −1 and 0.033 µg mL −1 .The suggested approach was successfully utilized for the quantification of ACA in laboratory-constituted dose formulation with a mean recovery of 100.19 ± 0.72% with no interference from its additives.Furthermore, the suggested approach was expanded to measure ACA in biological fluids.
A spectrochemical method was used to evaluate L-ORN in its pure state and as a nutritional supplement.Derivatization of L-ORN was accomplished with NBD-Cl through a nucleophilic substitution process (Figure 2) [58].The suggested method was effectively used on the L-ORN dietary supplements, indicating that additives and excipients did not interact.Through its reaction with the chemical NBD-Cl, αdifluoromethylornithine (DFMO) is determined spectrophotometrically and spectrofluorimetrically (Figure 3) [59].The optimal diluting solvent, heating time, temperature, and medium pH were optimized.The reaction product was evaluated spectrophotometrically at λmax = 478 nm and spectrofluorimetrically at λ emission = 540 nm after excitation = 475 nm.The spectrophotometric and the spectrofluorimetric methods were linear over 5-30 µg mL −1 and 0.4-2 µg mL −1 , respectively.The detection limits for the spectrophotometric technique and the spectrofluorimetric method, respectively, were 0.90 µg mL −1 and 0.071 µg mL −1 , making the suggested study an acceptable sensitive and selective measurement for DFMO.Additionally, both approaches had satisfactory recovery findings for DFMO in pharmaceutical cream samples.

Ninhydrin as derivatizing agent
Ninhydrin is the most popular substance for detecting latent fingerprints on permeable materials like paper and cardboard.NIN interacts with the amino acid (eccrine) component to produce Ruhemann's purple (a dark purple product) [60].The regulation of development factors like temperature, acidity (pH), and humidity are highly desired because the chemical reactions involved are complicated.Specifically, the amino acid analysis is performed using NIN in many bioanalytical methods."Ruhemann's purple" is created when ninhydrin interacts with the α-amino group of amino acids.For most amino acids, the same chromophore is produced [61].The intensity of the colour produced is determined by the quantity and chemical composition of the amino groups under investigation.The overall pH for the process is 5.5.Owing to the structural features of ninhydrin (NIN, Figure 4), it is possibly applied as derivatizing agent for the quantitative analysis of amino acids and imino acids.In a basic medium, NIN is transformed to o-carboxy phenyl glyoxal which further reduces NIN to 2-hydroxyindan-1,3-dione (HID).Analytes containing 1 o -NH 2 groups can react with HID to another compound that is on condensation with NIN to yield diketohydrindylidene-diketohydrindamine (II) [62].NIN undergoes tautomerization to 1,2,3-indantrione, which combines with the amino acid to produce a Schiff's base.The generated ketimine is decarboxylated, giving the aldehyde an intermediary amine.This intermediary amine is then condensed with a second NIN molecule to generate a purple product [63].
Wani et al. established a spectrophotometric process for the assessment of amino group-containing analyte cefixime (CFM) using ninhydrin as DA [64].Cefixime trihydrate (Figure 4) is a third-generation cephalosporin antibacterial agent that is taken orally and is often utilized to cure gonorrhea, tonsillitis, and pharyngitis [65].The method involves a reaction between the 1 o -NH 2 group of CFM with NIN in a basic medium to generate a yellow product that has λmax at 436 nm.It was observed that 1 mL of 0.2% w/v NIN at a temperature of 80 o C was optimum for a successful product with CFM.The molar ratio of CFM and NIN was found to be 20:1 (NIN: CFM) sufficient to develop a colour, however, at molar ratios of 100:1, the reaction remains unaffected.A lower molar ratio of 10:1 was not suitable for an acceptable colour generation.It was suggested that not ideal stoichiometry in the complexation between ninhydrin and CFM and thus interfering colour.

p-dimethylamino benzaldehyde as derivatizing agent
The compounds containing an amino group that does not possess chromophoric moiety can be evaluated using the spectrophotometric method through an appropriate reagent that contains carbonyl moiety.For instance, Patil and Wani reported a spectrometric approach for the estimation of pregabalin (PGB) from bulk and the capsule formulation using p-dimethylamino benzaldehyde (PDAB, Figure 6) [67].PGB (Figure 3), also known as Lyrica (S)-[ + ]−3-isobutyl GABA or (S)−3-(aminomethyl)−5-methyl hexanoic acid, is an anticonvulsant and analgesic prescription drug that is structurally and biologically active associated to gabapentin [68].
The reaction between the amino group of PGB with -C = O of PDAB in an acidic medium yielded a PGB-DAB complex which displayed absorption maxima at 395.80 nm (Figure 3).Regarding statistical evaluation, the t-value and F-value are computed and determined to be 0.60 and 0.08, respectively.The suggested approach could be used for regular quality assurance evaluation of PGB in dose form.It was observed that a molar ratio of 4:1 (PDAB: PGB) was adequate for optimum colour development.A calibration plot was constructed for PGB and displayed a linearity range between 5-60 µg mL −1 with r 2 = 0.9960.The LOD and LOQ were determined as 0.025 µg mL −1 and 0.076 µg mL −1 , respectively.
The novel technique relies on the creation of a yellow product with PDAB, followed by absorbance detection at 410 nm for determining olanzapine (OLP) in pharmaceuticals [69].This novel technique relies on forming a yellow reaction product with PDAB, followed by absorbance detection at 410 nm (Figure 7).At 50 °C and 10 min, the reaction factors were adjusted.The process took place with a stoichiometric ratio of 1:1.Absorbance was discovered to rise linearly with drug concentration  Reproduced from ref [69].
and served as the foundation for measurement.The calibration curve showed a linear relationship between 5 and 160 µg mL −1 , with a correlation value of 0.999.The observed molar absorptivity was 0.6103 L mol −1 cm −1 , and the Sandell sensitivity was assessed to be 49.50 ng cm −2 .The detection and quantitative levels were 6.6 and 20 µg mL −1 , respectively.The technique was verified in terms of precision, accuracy, and reproducibility.The recovery rate ranged from 98.4-101.5%,with a mistake rate of less than 1.7%.The suggested technique was tested for accuracy and precision against the standard Indian Pharmacopoeia high-performance liquid chromatography approach when examining OLP in pure and dosage formulations.Excipients that are widely used did not cause any conflict.The technique is easily adaptable for use in developing nations lacking advanced apparatus.

Pyrogallol as derivatizing agent
Many of these organic reagents have received a lot of interest because they are sensitive, colorimetric reagents as well as intriguing complex-forming reagents.Azo compounds containing the -N = N component are amongst the most well-researched groups of DAs, both theoretically and practically.Because aromatic derivatives contain an azo bond, they are extremely useful in the dyestuff business, colorimetry, and pharmacology [70][71][72].Pyrogallol (PGL, Figure 8) is also known as 1,2,3-Trihydroxybenzene (Figure 4).Sulfadiazine is a sulfonamide antibacterial agent that is utilized to treat several infectious diseases, including bladder infections, trachoma, and chancroid.
Numerous varieties of a specific organism, nevertheless, may be vulnerable.Sulphonamides suppress bacterial amplification by competing with p-aminobenzoic acid in the folic acid metabolism cycle.Microbial responsiveness to the numerous sulphonamides is the same, and resistance to one sulphonamide implies resistance to all.Most sulphonamides are easily absorbed orally.Sulfadiazine inhibits the microbial enzyme dihydropteroate synthetase competitively.This enzyme is required for the appropriate handling of PABA, which is considered necessary for synthesizing folic acid.The inhibitory effect interaction is required to produce folic acid in these microbes.
Naser and co-workers reported a method that involves the derivatization of sulfadiazine (SUL) with PGL, to yield 4-(4-sulphophenylazo) [4-SPAP] (Figure 8) [73].The coupling of pyrogallol with diazotized SUL at regulated pH.The solvo-chromic behaviour of 4-SPAP in various solvents of variable polarities was also examined.The derivative was then analyzed spectrophotometrically by initial rate and fixed time processes.These approaches involve the interaction of 4-SPAP with Ca (II) to yield a coloured derivative with an e max at 520 nm.The calibration graphs were plotted for two approaches indicating that the methods were linear in the range 1 × 10 −5 -2 × 10 −4 M. The LOD and LOQ were observed to be 0.35 and 1.05 µg mL −1 , respectively, using the fixed time method.
The presence of typical excipients in the pharmaceutical preparations had no discernible effect.The quantitative assessment of the suggested techniques with the standard reference was revealed, with great consistency and no substantial variation in their accuracy and precision evidenced by the F-and t-test statistics.The stoichiometric ratio of the interaction (4-SPAP-Ca(II)) was investigated using the molar ratio technique [74].To determine the complex stoichiometric ratio, a mole ratio technique was performed at 1 × 10 −3 M solutions of both reagent and metal salt.A series of solutions were prepared in which the reagent concentration was held fixed while the metal ion concentration varied, and the optimal conditions were established at the λmax for the derivative, the absorbance of the solutions was measured versus blank and plotted versus the mole ratio.At 520 nm, the stoichiometric ratio of 4-SPAP to Ca (II) was determined to be 1:1.Moreover, the proposed technique of measurement necessitates the use of basic apparatus that is readily accessible in any analytical laboratory.All the characteristics indicate that the suggested method is beneficial and suitable for quality assurance and regular measurement of the investigated substance in pharmaceutical dosage forms.More research is needed to evaluate other sulfadiazine-containing drugs, such as tablets and gauze.

9,10-phenanthraquinone (PQ) as derivatizing agent
9,10-phenanthraquinone (PQ), Figure 5, contains reactive keto groups and is used as a fluorescent agent for the identification of many organic compounds.The reactive keto act as the highly electrophilic centre which is suitable for reaction with compounds containing nucleophilic groups like -NH 2 , -OH, etc. PQ has been employed as a derivatizing agent for the estimation of guanidine, mono-substituted guanidines, and biguanides [41].The methods developed involve a reaction of the guanidino group with PQ producing stable fluorescent derivatives.
When 9,10-phenanthrenequinone (PQ) is illuminated with a white-light LED (430 nm), it produces an excited state (PQ * ) which undergoes the [4 + 2] reaction of cycloaddition of luminous alkenes with electronrich alkenes such as vinyl ethers [75].Alkynes and alkenes, whether regular or electron-deficient, do not exhibit any union with PQ * .This bio-orthogonal ligation is selective and can be utilized for vinyl ethermodified cetuximab live cell imaging and orthogonal bioorthogonal protein staining in a time-dependent way.Now, other light sources cannot activate phenanthrenequinones. Phenanthrenequinone modification promises hope for red-shifted action.The suggested process is focused on evaluating the FI of GLB at λemission = 348 nm upon excitation at λexcitation = 226 nm and analyzing the FI of the derivative produced by chemical modification of MFH in alkaline media utilizing PQ (Figure 9) at λemission = 416 nm after at λexcitation = 240 nm.The suggested spectrometric approach allows for accurate drug measurement, with limits of quantification of 0.04 and 0.01 µg mL −1 for MFH and GLB, respectively, offering better sensitivity than the previously published approach.
Further, the FI was measured across the range of 0.1-0.5 mmol HCl added to the basic reaction mixture, and the findings revealed that a neutral or acidic medium generated better FI than in basic conditions, therefore 0.1 M HCl was employed to neutralize NaOH and make the medium acidic.It was observed that at room temperature, the fluorescence occurred within fifteen minutes and achieved maxima at 1.5 h which further remained stable up to 3 h; whereas at 50 °C, the fluorescence maxima were achieved in 20 min and remained stable up to 1 h.

Fluorescamine reagent
Fluorescamine (FLU, Figure 10) is an analytical reagent commonly employed for detecting primary amines.It reacts instantaneously with an amine group containing compounds at room temperature in aqueous media.The derivatives are extremely fluorescent, while the reagent and its metabolites are non-fluorescent [79].Fluorescamine is used for more accurate measurement of amino acids using fluorescence spectroscopy [80].The chemical is typically dissolved in acetone because it rapidly hydrolyzes in water solutions.It is frequently used to identify amino acids on surfaces like TLC plates (in thin-layer chromatography).Amino acids can be identified by measuring the absorption of light at 470 nm after stimulation at 390 nm with detection levels in the low picomole region.
Recently, Omar et al. reported spectrofluorimetric determination of tranexamic acid (TXA) via its derivatization with FLU in the basic medium (Figure 10) [81].The reaction between TXA and FLU resulted in a highly fluorescent material (Figure 6) that shows fluorescence maxima at 473.5 nm after an excitation of 392 nm.It was noted that the molar ratio of TXA to FLU was 1:1 adequate for reaction in the presence of borate buffer at pH 8.3.Validation studies performed showed that the method gives a linear relationship in the concentration range of 0.1-0.9µg mL −1 .The LOD and LOQ values were reported as 0.0237 and 0.0719 mg mL −1 , respectively.Furthermore, the method was investigated for its  application to an in-vitro study spiking TXA in human plasma with a mean recovery of 99.430 ± 0.623%.
The earlier same group investigated the use of FLU as DA for the spectrometric estimation of doxazosin mesylate (DOX, Figure 11) in pure form, human plasma, and dosage formulations [81].This method involved a reaction (Figure 11) between DOX and FLU in a buffer solution (pH 3) that yielded an extremely fluorescent derivative observed at 489 nm via an excitation wavelength of 385 nm.The calibration plot displayed a linear relationship between concentration and absorbance in the range of 16-400 ng mL −1 .The method resulted in a LOQ value of 14.3 ng mL −1 , which indicated that the method provides nano-level sensitivity.
process between LND and FLU to create an extremely fluorescent product; which was recorded at 494 nm after being excited at 381 nm (Figure 12).Under the optimum conditions, a linear relationship between the fluorescence intensity and the LND concentration in the range of 25-300 ng mL −1 was discovered, with an excellent r 2 = 0.9999.The LOD and LOQ were 2.9 and 8.7 ng mL −1 , respectively.The RSD values were less than 1.4% for the method.The suggested technique was effectively applied for measuring LND in its bulk form and pharmaceutical capsule forms with acceptable precision; the recovery values were 97.8-101.4± 1.08-2.75%.The suggested approach is straightforward and found to be beneficial in determining LND.

O-phthalaldehyde/2-mercaptoethanol (OPA-MCE) as derivatizing agent
The synthesis of the isoindole derivative was analyzed at various time intervals, as well as the stability of the established reaction by examining the stability of FI over time up to 3 h [83].The reaction was completed within twenty-five minutes, and the derivative obtained was found to be stable for 180 min.α-Difluoromethylornithine, commonly known as Eflornithine (EFL, Figure 13), is a food and drug administration (FDA) recommended medicine that is employed to cure sleeping sickness as well as to reduce undesired facial hair in hirsutism.The suggested research is established on the formation of an extremely fluorescent isoindole product by condensing the amino component of EFL and OPA-MCE (Figure 13).The fluorescence and Resonance Rayleigh Scattering (RRS) intensities of the derivative were increased by 153% and 250%, correspondingly, when hexadecyl-trimethyl ammonium bromide was added.
The generated isoindole compound was fluorometrically determined at 429 nm after 337 nm after the reaction parameters were optimized.Furthermore, a considerable increase in the produced product's RRS intensity was recorded at 422 nm.The suggested approaches were verified following ICH requirements in terms of accuracy, sensitivity, robustness, and precision.In addition, the suggested methodologies were effectively used to test EFL in several commercially available brands of medicinal cream specimens with good recovery.Furthermore, the present fluorometric method was found to be effective in the assessment of EFL in spiked plasma and urine samples with excellent recovery.
EFL was calorimetrically assessed in a few colorimetric techniques depending on the availability of the 1 o -NH 2 group in their framework, either by chemical modification with PDAB, dansyl chloride, vanillin sodium, and 1,2-naphthoquinone-4-sulfonate (NQS) reagents or by an ion-pair complexation with methyl orange (MO), bromothymol blue (MB) [84,85].All the spectrometric techniques discussed above were restricted to determining Eflornithine hydrochloride in pharmaceutical vials and pharmaceutical formulations solely, with no application to pharmacological cream specimens or biologically active compounds.
HTA-Br was used as green fluorescent and RRS enhancer to create a novel spectrofluorometric technique recorded at λemission 429 nm and the RRS method measured at λmax 422 nm.Because the condensation interaction among EFL and OPA causes a significant "Turn on" in Eflornithine's fluorescence output.The condensation process causes a little augmentation in the RRS spectrum, whereas the micellar media generates a significant improvement.As a result, the spectrofluorometric approach was used to carefully optimize reaction parameters like reaction time, the amount of OPA, pH, the amount of buffer, and volume of 2mercaptoethanol, variety of surfactants, and type of diluting solvents.The amount of HTA-Br was studied using fluorometric and RRS techniques due to the significant influence of HTA-Br on the RRS spectra of the generated isoindole product.

HCl for derivatization
Chemically, hydrochloric acid (HCl) acts as a source of proton donors for acid-catalyzed reactions.The goal of this study was to establish and verify a simple, accurate, precise, and fast ratio first-order derivative spectrometric technique for estimating artemether (ART) and lumefantrine (LUM) simultaneously in a constant dosage tablet formulation [86].Because ART does not absorb UV light, the initial stage in developing the process was to derivatize it utilizing hydrochloric acid as the derivatizing agent.The derivatization procedures were further adjusted using a complete factorial multivariate method, with the amount of conc.HCl and the time needed for derivatization of ART at room temperature as independent variables (Figure 14).Furthermore, derivatizing parameters were adjusted relying on empirical investigation, i.e. 1.3 mL of conc.HCl at room temperature for 30 min.ART was determined by UV detection under these conditions, but the absorbance of LUM was reported to be unchanged.The devised approach yielded satisfactory calibration results for ART in the range of 5-30 µg mL −1 and LUM in the range of 2-12 µg mL −1 .The mean percent recovery values for ART and LUM were determined to be 99.96-100.49percent and 99.48-100.31percent, respectively.Furthermore, the developed approach was successfully employed in the estimate of ART and LUM in commercialized tablets, indicating that it may be used for quality assurance in the pharmaceutical industry.

NQS as derivatizing agent
Recently, Ali and co-workers developed two spectrometric methodologies for the estimation of monoso dium glutamate (MSG, Figure 15) [87].The two methods involved the derivatization of MSG with 1,2naphthoquinone-4-sulfonate sodium (NQS, Figure 15) (in the case of method I) and ascorbic acid (AA) (in case of method II) to yield coloured substances.The developed methods were optimized in terms of factors influencing reaction, one factorial at a time and the complete factorial method was employed in a method I and method II, respectively.It was found that Beer's law held good in ranges of 5-35 µg mL −1 and 1-14 µg mL −1 for methods I and II, respectively.Utilizing an analytical Eco-scale, the approaches were found to have good greenness.The findings led to suggestions regarding MSG's safety margin and persons who could be in danger.The suggested methodologies can be used to measure MSG in food as bromatological instruments.The procedures given are quick, straightforward, and costeffective, making them ideal for regular MSG testing in food and medicines.

bromate-bromide mixture as the derivatizing agent
Many dyes are permanently degraded to colourless substances in acidic media by oxidizing using agents.This process was used to determine several medicinal substances through indirect visibility spectrophotometrically.Recently, acidified solutions of the bromate-bromide mixture (BBM, KBrO 3 -KBr) and dyes have been utilized   as DAs for the quantitative determination of API [88].Kumar et al. developed two procedures involving the bromination of alogliptin (AGN) in an acidic medium with a standard BBM composition [89].This is supported by a quantitative evaluation of excess bromine to bleach MO (method A) or MB (method B).Br 2 is produced in situ by the reaction of HCl on the BBM (Figure 16).The calculated excessive Br 2 will assist in bromination AGN, and the remaining Br 2 reacts with a predetermined quantity of MO or MB.The quantity of Br 2 reacted with MO or MB correlates to the quantity of AGN and thus serves as the basis for AGN assays using procedures methods A and B.
When MO (method A) and MB (method B) are bleached with Br 2 , the absorbance at 505 and 720 nm reduces and approaches the lowest level.When the concentration of AGN increases, the concentration of bromine decreases.It can be demonstrated by a linear rise in absorbance caused by unbleached MO (method A) and MB (method B) at 505 and 720 nm, respectively.It was observed that 10 mg mL −1 KBrO 3 produces the highest and most reproducible absorbance in method A and 20 mg mL −1 KBrO 3 produces the highest and most reproducible absorbance in method B. As the quantity of KBrO 3 increased further, the absorbance value gradually decreased.As a result, KBrO 3 -KBr mixed solutions containing 10 µg mL −1 KBrO 3 and 20 µg mL −1 KBrO 3 were chosen for methods A and B, respectively.With 2 mL of 5 M HCl, the maximum absorbance was achieved.
At a MO concentration of 50 µg mL −1 , the maximum absorption intensity was obtained.Increased MO concentrations up to 90 µg mL −1 showed no influence on absorption values.The maximum absorbance value was obtained at a concentration of 40 µg mL −1 MB, after which the absorbance value decreased significantly.The validation parameters were determined to be suitable for methods A and B for concentration ranges of 1-10 µg mL −1 and 2.5-12.5 µg mL −1 , respectively.The detection limits for methods A and B were 0.115 µg mL −1 and 0.210 µg mL −1 , respectively.Recovery experiments were used to evaluate the accuracy of the suggested techniques, and the percentage recoveries of AGN were determined to be greater than 99% for both methods A and B.

Charge transfer complexes
A charge-transfer complex is formed when the electronic charge is transferred from an "electron-rich" component to an "electron-deficient" component.Consequently, one component acquires somewhat positively charged with the other, forming a weak ionic association.The production of bright-coloured chargetransfer (CT) complexes, that absorb visible radiation, is often related to molecular associations among electron donors and electron acceptors.Because charge transfer complexes develop quickly, they may be used to construct easy and practical spectrofluorimetric techniques for a variety of medicinal drugs that act as electron donors [90][91][92][93].As a weak acid salt and a negatively charged carrier, sodium valproate (SV) is an excellent electron donor and may produce CT complexes with a variety of acceptors.
The identical technique was applied to comparable weak acid salts, including losartan potassium (LP) and rabeprazole sodium (RS), that interacted successfully with a variety of electron acceptors, and the coloured derivatives were used in spectrometric studies [94].The notion that the charge transport complex formation processes of VP have yet to be addressed owing to the lack of analytical literature reports inspired us to create easy, quick, and accurate spectrometric techniques for the evaluation of VP for quality assurance reasons.These simple techniques of examination are vital and effective, particularly for medications that lack chromophores and, as a result, lack spectrometric analytical procedures.These approaches suggested in this report are built on charge transport connections among VP as an electron donor and p-chloranilic acid (CA), dichlone, picric acid (PA) as π -acceptors, and iodine as σ -acceptor.
SV is an excellent electron donor and may generate CT complexes with a variety of acceptors since it is a weak acid salt and a negative charge carrier.Similarly, weak acid salts, including LP and RS, which interacted efficiently with numerous electron acceptors, were treated in the same way, and the colourful products were used in spectrophotometric tests [95][96][97].Because no quantitative data on the charge transfer complex formation processes of VP have been identified in the literature, we were inspired to create simple, quick, and accurate spectrometric techniques for the assessment of VP for quality assurance reasons.These easy and fast techniques of assessment are particularly significant and effective in the context of medications that lack fluorescent dyes and, as a result, lack spectrofluorimetric diagnostic procedures.
The approaches suggested being built on CT interactions among VP as an electron donor and e-acceptors such as p-CA, dichlone, and PA, as well as iodine.The CT complex formation processes of SV as an electron donor with a variety of electron acceptors were studied (Figure 17).The colourful complexes produced were used to build four novels, simple, quick, and efficient spectrofluorimetric techniques for analyzing sodium valproate in pure form and dose forms.According to a review of the literature, most sodium valproate analytical techniques are dependent on HPLC, which is generally connected with chemical modification.
The reaction involves the proton transfer from the PA to AGN at room temperature to yield a yellow-coloured ion-pair complex in a CHCl 3 (Figure 18).The obtained yellow-coloured complexes displayed λmax at 415 nm.Validation results of the two methods indicated a linear response in the concentration range of 10-60 µg mL −1 .It was also noted that there was no interference due to common tablet excipients.Stoichiometric analysis carried out using Job's process of continuous variation showed that a 1:1 ratio of AGN and PA was required to form an ion-pair complex for both methods.The introduction of 1.0 ml of 0.4% PA solution was adequate to get the highest and most consistent absorbance readings.
Belal and co-workers developed direct spectrophotometric approaches for the assessment of SV via CT complex formation processes [104].The approach involves the reaction of SV and PA in chloroform forming a yellow product that showed e max at 415 nm.The calibration curves of the generated colour derivatives with 2-20 µg mL −1 .Considering excellent accuracy and precision, the suggested methodologies were satisfactorily used in the assessment of SV in tablets and oral solution drug products.The assay outcomes were statistically evaluated to a standard active pharmaceutical HPLC technique, and no considerable irregularities between the suggested approaches and the reference method were found.Stochiometric analysis performed using Job's method approached a maximum value at a mole fraction of 0.5 for interactions of VP with PA demonstrating a molar ratio of 1:1 for VP to PA.
Baker et al. used PA for the estimation of maduramicin ammonium (MAD) using spectrophotometricbased methods [105].Since MAD does not possess any chromophoric moiety, difficult to analyze through UV-Vis spectrophotometry.The method is based on complex formation between MAD with PA which was measured at 405 nm.The validation results demonstrated that the three methods were observed in the linear concentration range of 30-150 µg/mL.The methodology was effectively applied in the quantitation of MAD drug formulations for veterinarian usage.
2,4-dinitrophenol (DNPh) is employed as an analytical reagent for the measurement of pharmaceutically significant compounds with primary or secondary amino groups in their architecture [106,107].Kumar et al. studied the interaction of AGN with DNPh to determine the alogliptin (AGN) in bulk and tablet formulations [101].The reaction involves the proton transfer from DNP to AGN at room temperature to yield a yellowcoloured ion-pair complex in a CHCl 3 (Figure 13).The obtained yellow-coloured complex displayed λmax at 430 nm.Validation results of the method indicated a linear response in the concentration range of 10-50 µg mL −1 .Further, it was also noted that there was no interference due to common tablet excipients.Stoichiometric analysis carried out using Job's process of continuous variation showed that a 1:1 ratio of AGN and DNP was required to form an ion-pair complex for both methods.The introduction of 1.0 ml 0.1% DNP solution was adequate to get the highest and most consistent absorbance readings.

p-chloranilic acid (p-CA) as compelxing agent
p-CA is a neutral molecule with localized, clearly characterized single and double bonds, while monoanions have a delocalized π -system comprising half of the rings.The dianion has two delocalized pi-systems separated by two single C-C bonds.It functions as a piacceptor.Belal and co-workers developed direct spectrophotometric approaches for the assessment of SV via CT complex formation processes [78].The first approach involves a reaction of the SV with p-CA in acetone to offer a purple-coloured derivative with e max at 524 nm.The calibration curve of the generated colour derivatives with p-CA demonstrated strong linear relationships across concentration levels of 24-144 µg mL −1 , correspondingly.Considering excellent accuracy and precision, the suggested methodologies were satisfactorily used in the assessment of VP in tablets and oral solution drug products.Stochiometric analysis performed approached a maximum value at a mole fraction of 0.5 for interactions of PA with p-CA.
Baker et al. used p-CA for the estimation of MAD using spectrophotometric-based methods [105].MAD (Figure 19) is a polyether carboxylic ionophore substance approved for use as a coccidiostat feed additive in chicken and turkey to suppress E. adenoides, E. meleagrimitis, E. gallopavonis, and E. dispersa.Since MAD does not possess any chromophoric moiety, difficult to analyze through UV-Vis spectrophotometry.The method is based on complex formation between MAD with p-CA which was measured at 519 nm.The validation results demonstrated that the method was a linear concentration range of 100-1000 µg mL −1 .Adegoke et al. developed a simple, precise, and exact concurrent spectrometric technique for evaluating trimethoprim-sulphamethoxazole combinations in pure and tablet dose formulations [108].Sulphonami des (SMZ) were the first efficient chemotherapeutic drugs used extensively in the preventive measures and treatment of bacterial infections in humans [109].Trimethoprim (TMP) is a typically combined agent.The combination is employed to address urinary tract infections as well as a potent bactericidal drug [110].
Trimethoprim (TMP) is complexed with chloranilic acid (CAA) through direct charge transfer in an acetonitrile-dioxane solvent combination and SMZ is complexed following hydrolysis in dilute H 2 SO 4 (Figure 15).Temperature and time optimizations found that room temperature and 20 and 30 min, correspondingly, were optimal for TMP and SMZ.Because optimal detector responses were achieved at 520 and 440 nm, these wavelengths were chosen as operating wavelength maxima for SMZ and TMP, correspondingly.TMP and hydrolyzed SMZ were mixed with CAA in 1:1 and 1:3 mol ratios, correspondingly.On a three-day evaluation, accuracies were typically less than 4% (measured as the degree of inaccuracy or error), with a precision of the order of less than 2%.The physicochemical parameters that contribute to complicated stability were evaluated and correlated with the obtained data.Having accuracies equivalent to the standard BP technique, the approach was effectively utilized in the assessment of TMP and SMZ in tablet formulations.There have been no interferences from typical tablet excipients, and the TMP compound did not affect the SMZ test.The described approach might be used for routine analysis of the TMP-SMZ mixture Figures 20 and 21.

Dichlone as complexing agents
Belal and co-workers developed direct spectrophotometric approaches for assessing SV via CT complex formation processes [104].SV (Figure 16), often referred to chemically as sodium 2-propyl pentanoate, is an antiepileptic medication utilized to cure primary general seizures, absence, myoclonic seizures, and partial seizures.The approach involves the formation of an orange-red derivative by the reaction of SV with dichlone (DC) in DMF, which showed e max at 490 nm.The calibration curve of the generated colour derivative with DC demonstrated strong linear relationships across concentration levels of 40-200 mg mL −1 .Considering excellent accuracy and precision, the suggested methodologies were satisfactorily used in assessing VP in tablets and oral solution drug products.Stochiometric analysis revealed that a molar ratio of 1:1 for SV to DC was adequate to create a complex at a mole fraction of 0.5 for interactions of PA with DC.

2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ)
Baker et al. used three derivatizing agents viz.p-chloranilic acid (p-CAA), 2,3-dichloro-5,6-dicyano-pbenzoquinone (DDQ), and picric acid (PA) for the estimation of Maduramicin ammonium (MAD) using spectrophotometric based methods [105].Since MAD does not possess any chromophoric moiety, difficult to analyze through UV-vis spectrophotometry.The developed method was based on the complexation of MAD with p-CA/DDQ/PA; the complexes were examined at 519, 588, and 405 nm.The validation results demonstrated that the three methods were observed in the linear concentration range of 100-1000, 25-250, and 30-150 µg mL −1 .The methodologies were effectively applied for estimating MAD drug formulations for veterinarian usage.Table 1 indicates the analytical performance of derivatizing agents for the spectrometric determination of various pharmaceuticals.

Conclusion and future prospective
Over the decades, the scientific literature has undergone significant growth in the number of derivatization techniques, and additional agents are being released periodically for the evaluation of prescription drugs.Novel reagents are routinely described in all of these classifications, as are possible uses for existing agents.The enhanced sensitivities, specificity, and broad applicability of chemical derivatization methods are significant benefits.Because most of the techniques involve specified processes, the sample amounts to be measured are frequently in the microgram range.Chemical derivatizing agents frequently take up analytes depending on definite chemical concepts; the processes could be very selective, and they can be modified by changing the solvent, pH, or other essential experimental parameters.Another excellent benefit of chemical derivatization techniques is their broad application since several of the processes may be easily adjusted to meet reaction moiety.One significant objection raised against most derivatization methods is the usage of multistep phases in their formulation and deployment.Because of the rising expense of pharmaceutical formulations when advanced procedures are used, the importance of derivatization approaches will grow in the coming decades.Modern pharmaceuticals, on the  Linearity: 0.1-0.9μg mL −1 LOD: 0.0237 μg mL −1 LOQ: 0.0719 μg mL −1 Mean recovery: 99.430 ± 0.623%.
Abdelrahman et al. reported a spectrofluorometric technique for determining Metformin HCl (MFH) and Glibenclamide (GLB) in a binary combination with no pre-resolution [76].The recently completed trial indicated that metformin reduces muscular hypertrophy by responding to gradual resistance exercise training in older individuals, implying that the potential advantage of MFH on longevity may not even translate to an advantage in all tissues.Glibenclamide (GLB), or Glyburide (Figure 9), is a chemical compound with the molecular formula 1-[[4-[2-[(5-Chloro-2-methoxy benzoyl)amino]ethyl]phenyl]sulphonyl]−3-cyclohexyl urea and utilized to cure type 2 diabetes mellitus and can be administered safely by the fasting sick people with non-insulin-dependent diabetes [77,78].

Figure 15 .
Figure 15.(i) Derivatization of MSG with NQS (in case of method I) and AA (in case of method II) to yield coloured substances, (ii) UV/Vis absorption spectra of; (a) the reaction product of MSG and NQS against reagent blank, (b) NQS against blank, (iii) (c) the reaction product of MSG and AA against reagent blank, (d) AA against DMF.Reproduced from ref [87].

Figure 16 .
Figure 16.(a) Derivatization of AGN with BBM and MB (a), absorption spectra of coloured product in method A (b) and method D (d), the effect of concentration of KBrO3 (c) and HCl (e).Reproduced from ref [89].

Figure 20 .
Figure 20.Complexation between TMP with CAA and SMZ through direct charge transfer.Reproduced from ref [108].

Table 1 .
Continued.otherhand, are replete with approaches ing some of these costly techniques, with derivatization processes now retained for simple test-tube reactions for identification.Derivatization procedures, on the other hand, are expected to remain useful for the quality assurance of the bulk of medicines.The possibility of novel reagents being introduced shortly cannot be ruled out.It is possible to carry out such derivatization methods under needs and experimental circumstances thanks to the development of newer reagents from several derivatization reagent forms, which may allow the change of a specific feature of relevance.