Ninhydrin-Mediated Spectrophotometric Assay for the Simultaneous Analysis of Pregabalin and Duloxetine in Pharmaceutical Mixtures

2.1. Instrumentation

All absorbance measurements were performed using a Shimadzu UV-1900i PC double-beam UV-Visible spectrophotometer (Shimadzu Corporation, Japan), equipped with 1.0 cm path length quartz cuvettes. Spectral data were acquired and processed using Labsolutions UV-VIS software. A JENWAY pH metre model 3510 (Cole-Parmer, UK) was used for pH measurements when required. Sample dissolution and homogenization were facilitated by an ultrasonic bath (FALC Instruments, Italy). An analytical balance (Vibra HT analytical balance, Japan) with a precision of ±0.01 mg was used for accurate weighing of standards and reagents.

2.2. Chemicals and reagents

PG and DL hydrochloride (purity >99.8%) were obtained as gift samples from EVA PHARM (Cairo, Egypt) and were used as reference standards. Certificate-verified purities were 99.92% for PG and 99.80% for DL. Ninhydrin (purity 99.86%) was purchased from Loba Chemie Pvt. Ltd. (Mumbai, India). Methanol (HPLC grade) was acquired from Fisher Scientific (UK). All other chemicals and solvents were of analytical reagent grade. Freshly double-distilled water was used throughout the study for the preparation of all aqueous solutions and dilutions.

2.3. Preparation of standard solutions

Stock solutions (1000 µg/mL): Accurately weighed quantities of PG (50.0 mg) and DL (50.0 mg) were separately transferred into 50 mL volumetric flasks. Each compound was dissolved in approximately 25 mL of methanol, followed by sonication for 5 minutes to ensure complete solubilisation. The solutions were then diluted to the mark with methanol, resulting in stock solutions of 1000 µg/mL (1.0 mg/mL) for each drug. These stock solutions were stable for at least one week when stored in amber glass bottles at 4°C.

Working standard solutions (100 µg/mL): Aliquots of 1.0 mL from each stock solution were transferred into separate 10 mL volumetric flasks and diluted to volume with methanol to obtain working standard solutions of 100 µg/mL for both PG and DL. These dilutions were used for the preparation of calibration standards and synthetic mixtures.

Synthetic binary mixtures: To evaluate the accuracy, specificity, and applicability of the method for simultaneous analysis, synthetic binary mixtures of PG and DL were prepared in methanol at various concentration ratios within the linear range of the assay (e.g., 1:1, 1:2, 2:1). These laboratory-prepared mixtures simulated potential pharmaceutical combinations and were used to assess the absence of mutual interference between the two analytes under the developed analytical conditions.

2.4. Procedures

The development of the simultaneous spectrophotometric method for PG and DL was based on a dual-detection strategy that exploits the complementary spectroscopic behaviour of the two drugs. The fundamental principle involves the indirect quantification of PG through a chromogenic derivatisation reaction with ninhydrin, while DL is determined directly via its intrinsic UV absorbance, thereby enabling their concurrent analysis without physical or mathematical separation.

3.1. Spectral characteristics

The analytical challenge in the simultaneous determination of PG and DL arises from their fundamentally different molecular architectures and, consequently, their different spectroscopic behaviours. PG, chemically known as (S)-3-(aminomethyl)-5-methylhexanoic acid, is a lipophilic analog of γ-aminobutyric acid (GABA) that lacks aromatic rings or extended conjugated π-systems. As a result, its electronic transitions occur primarily in the far-UV region, where absorbance is weak, poorly resolved, and highly interfered with by common solvents and excipients [Figure 2]. This absence of a strong chromophore renders direct UV-Vis spectrophotometric quantification impractical, particularly in multi-component systems such as fixed-dose combinations or biological matrices where overlapping signals can compromise selectivity and accuracy.

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Figure 2:

Zero-order UV absorption spectrum of pregabalin in methanol (a) before and (b) after ninhydrin derivatisation. (c) Overlayed different concentrations of pregabalin-ninhydrin complex and Duloxetine HCl at different concentrations. The colour-coded curves represent the absorbance spectra of the pregabalin-ninhydrin complex at different concentrations. The colors correspond to increasing concentration levels, as follows: Highest concentration (Violet): 30 µg/mL, Red: 25 µg/mL, Black: 20 µg/mL, Blue: 15 µg/mL, Orange: 10 µg/mL, Blue: Lowest concentration (5 µg/mL), The increasing absorbance intensity with concentration demonstrates the method’s linearity and sensitivity for quantitative analysis.

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In contrast, DL (±)-N-methyl-3-(naphthalen-1-yloxy)-3-(thiophen-2-yl) propan-1-amine, contains a naphthalene ring, a polycyclic aromatic hydrocarbon, that serves as a robust chromophore due to its extended π-conjugation. This structural feature enables DL to exhibit a well-defined and intense absorption maximum at 230 nm, allowing for direct and reliable quantification via UV spectrophotometry without the need for chemical modification. The difference in detectability between the two drugs, where one is non-chromophoric and the other is inherently UV-active, presents both a challenge and an opportunity. The challenge lies in enabling the simultaneous analysis of both drugs without cross-interference, while the opportunity arises from exploiting this fundamental difference through a selective derivatisation strategy that targets only one component of the mixture. To bridge this analytical gap, a chemical derivatisation strategy was employed to enhance the detectability of PG. The key functional group targeted in this approach is the primary aliphatic amine located at the 3-aminomethyl position of PG. This primary amine is highly reactive toward ninhydrin (2,2-dihydroxy-1,3-indanedione), a classical chromogenic reagent extensively used in amino acid and amine analysis. The reaction between ninhydrin and primary amines is one of the most well-characterised transformations in analytical organic chemistry and forms the basis of the ninhydrin test, a standard qualitative and quantitative assay for α-amino acids and primary amines.^[15]

The reaction proceeds through a multi-step mechanism that begins with the nucleophilic attack of the primary amine of PG on one of the carbonyl groups of ninhydrin, forming a Schiff base (aldimine) intermediate. This step is followed by decarboxylation (in the case of α-amino acids) or direct dehydration (in the case of primary amines like PG), leading to the formation of a reduced ninhydrin species (dihydroruhemann’s purple) and an imine derivative. A second molecule of ninhydrin then condenses with this intermediate to yield the final product: Ruhemann’s purple (diketohydrindylidene-diketohydrindamine), a deeply coloured, resonance-stabilised quinoidal compound with a characteristic absorption maximum at 580 nm in the visible region.^[16] This chromogenic transformation is highly specific for primary amines, which are less sterically hindered and more nucleophilic than secondary or tertiary amines. The reaction does not proceed with tertiary amines, and secondary amines yield a different, yellow-coloured product, which absorbs at a shorter wavelength and does not interfere with the detection of Ruhemann’s purple.^[17] This selectivity ensures that only PG undergoes derivatisation, while DL, whose amine is tertiary (N-methyl group), remains unreacted and can be quantified directly at 230 nm. The complete spectral separation between the two detection wavelengths (580 nm for PG-N and 230 nm for DL) enables their simultaneous determination without the need for mathematical resolution, derivative spectroscopy, or physical separation techniques.

3.2. Method validation and statistical analysis

The proposed dual-detection method was rigourously validated according to the International Conference on Harmonisation (ICH) guidelines Q2(R1) to ensure its suitability for routine quality control applications. The validation parameters were evaluated for both PG and DL separately [Supplementary Table 1].

The linearity of the method was established over the concentration ranges of 3–30 µg/mL for PG [Figure 2b] and 1–35 µg/mL for DL [Figure 2c]. Calibration curves were constructed by plotting absorbance against concentration. The regression analysis yielded excellent correlation coefficients (r²) of 0.9996 for PG and 0.9997 for DL, indicating a strong linear relationship between the response and the analyte concentration within the specified ranges. The regression equations were determined as follows: For PG, Absorbance = 0.0371 C - 0.0068; for DL, Absorbance = 0.0207 C - 0.0138, where C is the concentration in µg/mL.

The accuracy of the method was assessed by performing recovery studies using the standard addition technique. Six different samples of each drug were prepared at three different concentrations and analysed in triplicate. The mean percentage recovery for PG was 100.57 ± 1.16%, while for DL, it was 100.58 ± 1.26%. These results confirm the high accuracy of the method, with recoveries falling within the acceptable range of 98–102%. The method’s precision was evaluated in terms of repeatability (intra-day precision) and intermediate precision (inter-day precision). Repeatability was determined by analysing six samples of each drug at three concentrations on the same day, yielding % RSD values of 1.15% for PG and 1.18% for DL. Intermediate precision was assessed by analysing the same samples on three successive days, resulting in % RSD values of 1.0742% for PG and 1.045% for DL. These low % RSD values demonstrate the high reproducibility and reliability of the method.

Specificity was confirmed by analysing a mixture of the two drugs, which showed no interference between the components, as evidenced by the distinct absorbance maxima at 580 nm for the derivatised PG complex and 230 nm for the native DL.

The LOD and LOQ were calculated using the formulae LOD = 3.3 × S.D./Slope and LOQ = 10 × S.D./Slope, respectively, where S.D. is the standard deviation of the response and Slope is the slope of the calibration curve. The LOD values were determined to be 0.5489 µg/mL for PG and 0.2425 µg/mL for DL. The LOQ values were 1.6633 µg/mL for PG and 0.7349 µg/mL for DL. These low limits of detection and quantitation indicate the high sensitivity of the method.

The method was further validated by applying it to laboratory-prepared mixtures containing different ratios of PG and DL [Supplementary Table 2]. The results demonstrated a mean percentage recovery of 100.34% for PG with a relative standard deviation (RSD) of 0.8794%, and a mean recovery of 99.7367% for DL with an RSD of 0.9538%. The low RSD values confirm the high precision of the method when applied to complex mixtures. The F-values for both drugs were significantly greater than the critical F-value at the 95% confidence level, indicating that the method is robust and reliable for the simultaneous determination of both drugs in binary mixtures.

3.3. Mechanistic and thermodynamic basis for optimisation of derivatisation conditions

The efficiency, reproducibility, and sensitivity of the ninhydrin-based derivatisation are profoundly influenced by the reaction environment, including solvent, temperature, time, pH, and reagent concentration. A deep understanding of the underlying organic chemistry is essential for rational optimisation.

The reaction mechanism involves polar intermediates and proton transfer steps [Figure 3], making it highly sensitive to solvent polarity and protic character. The figure shows the mechanism of the nucleophilic attack of pregabalin’s primary amine on the electrophilic carbonyl carbon of ninhydrin, forming a Schiff’s base via condensation and loss of two water molecules. This intermediate undergoes decarboxylation, releasing CO₂ and rearranging into a stabilised iminium species. A subsequent condensation with a second ninhydrin molecule, followed by elimination of water and 2-methylpentane-2,4-dione, yields the final Ruhemann’s purple chromophore. Each step involves transient polar species and proton transfer, which are strongly influenced by the solvent’s ability to donate and accept hydrogen bonds.^[18] Among the solvents evaluated, methanol, ethanol, acetonitrile, and water, ethanol emerged as the optimal medium. As a polar protic solvent, ethanol effectively solvates both ninhydrin and PG, facilitates proton transfer during condensation, and stabilises charged intermediates. It also enhances chromophore solubility, preventing precipitation and ensuring a homogeneous solution for accurate absorbance measurement. In contrast, aqueous media produced lower colour intensity due to limited ninhydrin solubility and potential reagent hydrolysis. Aprotic solvents like acetonitrile failed to support efficient reaction kinetics, underscoring the necessity of a protic environment for successful transformation.

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Figure 3:

The proposed mechanism of complex reaction of pregabalin and Ninhydrin.

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The concentration of ninhydrin was optimised to ensure stoichiometric excess for complete conversion of PG while minimising reagent waste and background absorbance. A 3% (w/v) ethanolic solution was found to be optimal. Lower concentrations led to incomplete derivatisation, as evidenced by sub-maximal absorbance, while higher concentrations did not enhance the signal and increased the risk of side reactions or reagent aggregation. This concentration aligns with those reported in classical methods for amino acid analysis, where a 2–4% ninhydrin solution is typically used to ensure quantitative reaction.^[19]

Temperature and reaction time are critical kinetic parameters. The formation of Ruhemann’s purple is an endothermic process that requires thermal activation to overcome the energy barrier for imine formation and subsequent condensation. Incubation at 100°C for 5 minutes was sufficient to achieve complete chromophore formation, as confirmed by a plateau in absorbance values. Prolonged heating beyond this point did not increase the signal and, in some cases, led to a slight decrease in absorbance, suggesting possible degradation of the chromophore or oxidative side reactions. The use of a thermoblock ensured uniform and reproducible heating, minimising variability between samples.

The pH of the reaction medium also plays a crucial role, although in this case, the reaction was carried out in ethanol without explicit pH adjustment. In aqueous systems, the ninhydrin reaction is known to proceed optimally under slightly acidic to neutral conditions (pH 5–7), where the amine is sufficiently nucleophilic (as the free base) while the carbonyl groups of ninhydrin remain electrophilic. In strongly acidic media, the amine is protonated and thus less nucleophilic, slowing the reaction. In strongly alkaline conditions, ninhydrin may undergo hydrolysis or side reactions. The use of ethanol as a solvent effectively buffers these effects, providing a neutral, non-aqueous environment conducive to the reaction.

3.4. Quantitative greenness outcomes and sustainability interpretation

The greenness assessment yielded quantitatively favourable outcomes across all four tools [Table 1]. The AGREE calculator returned a score of 0.75, derived from 12 criteria: 8 scored green (waste <10 mL/sample; energy = 5 min at 100°C + UV-Vis; ethanol as renewable solvent; no PBT reagents; simple sample prep; high throughput; biodegradable waste; operator safety under standard lab conditions), while 2 scored yellow (no real-time monitoring; manual operation), and one scored red. The GAPI pictogram showed five green segments, justified as follows: (1) sample collection involved solid tablets (no biohazard); (2) sample preparation required only 1 mL methanol dissolution + 1 mL 3% (w/v) ninhydrin in ethanol, with no extraction or cleanup; (3) reagents included ethanol (Class 3 solvent, ICH Q3C), ninhydrin (0.03 g per sample), and trace methanol (1 mL, Class 2); (4) instrumentation used a low-power UV-Vis spectrophotometer (<100 W); and (5) total waste was 8.3 g/sample (6.3 g ethanol + 0.8 g methanol + 0.03 g ninhydrin + 1.2 g water-equivalent), all biodegradable. The NEMI pictogram was fully white because: (i) no PBT substances were used (CAS 485-47-2 for ninhydrin is not PBT); (ii) no RCRA-listed waste was generated; (iii) reaction pH was neutral (∼7 in ethanol); and (iv) waste mass (8.3 g) was <17% of the 50 g threshold. Finally, the BAGI tool assigned a score of 82.5/100, calculated from: sample volume = 1 mL (max 5 mL allowed), preconcentration = not required, reagent hazard = low (ethanol dominant), energy = low (5 min heating), and instrumentation = non-hazardous. Complementing these results, the method achieved an excellent Eco-Scale score of 92, reflecting a total penalty of only 8 points assigned according to the reagent hazard, energy use, waste generation, and operator safety. Collectively, these metrics confirm the method’s high sustainability, with <10 mL solvent consumption, <9 g waste, and zero use of halogenated or persistent chemicals.

Table 1: Greenness of the proposed method using AGREE, GAPI, NEMI, Eco-scale and BAGI assessment tools.

Eco-scale assessment tools
Reagents parameter		Penalty points
Ethanol (solvent and diluent) Methanol (sample dissolution) Ninhydrin (3% w/v solution) Energy consumption Waste generated Operator safety		1 2 1 1 2 1
Total penalty Score		8 92
AGREE	GAPI	NEMI	BAGI