Green HPLC Method Delivers Fast, Precise Testing of Combination Depression Therapy

Researchers in India have developed and validated the first reported reversed-phase high-performance liquid chromatography method capable of simultaneously measuring brexpiprazole and escitalopram oxalate in a combined formulation, and they have shown that the procedure is not only fast and accurate but also environmentally responsible. The work, published in Discover Chemistry by Divya Solanki and Zarna Dedania, addresses a genuine analytical gap: although both drugs are widely prescribed for major depressive disorders, no HPLC method had previously been published for their joint determination in a single dosage form, and no marketed combination product yet exists. The new method therefore provides a foundation for quality control should such combination therapies reach the clinic or pharmacy shelf.

The two active ingredients occupy distinct pharmacological territories. Brexpiprazole, chemically 7-[4-[4-(1-benzothiophen-4-yl)piperazin-1-yl]butoxy]-1H-quinolin-2-one, is an atypical antipsychotic classified as a serotonin-dopamine activity modulator. It partially activates D2 dopamine and serotonin 1A receptors and binds strongly to serotonin, dopamine, and alpha-adrenergic receptors; the United States Food and Drug Administration approved it on July 10, 2015. Escitalopram oxalate, structurally (1S)-1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-3H-2-benzofuran-5-carbonitrile oxalate, belongs to the selective serotonin reuptake inhibitor class and treats depression and anxiety by increasing serotonergic activity in the brain. The combination of the two agents was approved in 2014 and has been studied in clinical trials for mood disorders, which makes reliable analytical tools for the pair increasingly important.

To build the method, the team used a Shimadzu semi-automatic liquid chromatograph equipped with a photodiode array detector and Win Chrome software, injecting samples through a microliter syringe onto a reversed-phase C18 column measuring 250 millimeters by 4.6 millimeters with five-micron particles. Raw materials were supplied as gift samples by Virdev Pharma of Palsana and Sun Pharmaceutical Industries of Panoli, and the researchers prepared a synthetic mixture mimicking a combined dosage form at a ratio of 40 micrograms per milliliter of brexpiprazole to 120 micrograms per milliliter of escitalopram oxalate. Because no commercial product exists, the synthetic mixture served as the practical test bed for the entire validation exercise.

Optimization of the chromatographic conditions revealed that a mobile phase of methanol and phosphate buffer, adjusted to pH 4.0 with orthophosphoric acid and mixed in a 55:45 volume ratio, delivered the best performance. Elution ran in isocratic mode at a flow rate of one milliliter per minute, with detection at 214 nanometers and a total run time of ten minutes. Under these conditions brexpiprazole eluted at 2.7 minutes and escitalopram at 7.0 minutes in the reported system suitability work, with symmetrical peak shapes, theoretical plate counts above 2000, and tailing factors at or below 2.0. The separation logic is rooted in the chemistry of the two molecules: both carry basic functional groups and exist predominantly in protonated form at pH 4.0, but brexpiprazole is considerably more lipophilic, with a log P of roughly 4.7 to 5.0 compared with 3.5 to 4.0 for escitalopram. That difference governs how strongly each analyte interacts with the nonpolar C18 stationary phase and therefore dictates the elution order.

Validation followed the International Council for Harmonization guideline Q2(R2), the current global standard for analytical procedure validation. Linearity was assessed at five concentration levels with six replicates each, spanning 0.5 to 3 micrograms per milliliter for brexpiprazole and 5 to 30 micrograms per milliliter for escitalopram oxalate. Calibration curves returned correlation coefficients exceeding 0.999 for both drugs, indicating an exceptionally straight relationship between peak area and concentration across the working ranges. Accuracy was confirmed through standard addition and recovery studies at 80, 100, and 120 percent spiking levels, with satisfactory recoveries demonstrating that common formulation excipients do not interfere with quantification.

Precision, the method’s ability to produce consistent results, was evaluated in both intraday and interday experiments. Three standard solutions of different concentrations were injected repeatedly within a single day and again across three consecutive days, and the relative standard deviation stayed below 2.0 percent throughout. Specificity was demonstrated by analyzing blank mobile phase and sample solutions, with no interfering peaks appearing at the retention times of either analyte. Sensitivity limits were calculated from the standard deviation of the response and the slope of the calibration curve using the ICH equations LOD equals 3.3 times sigma over S and LOQ equals 10 times sigma over S, yielding low detection and quantification limits that confirm the method’s suitability for trace-level work. Robustness testing deliberately introduced small variations in flow rate, mobile phase composition, pH, and detection wavelength, while ruggedness was probed through changes of analyst and column, with retention times remaining stable in all cases.

When the optimized method was applied to the synthetic mixture containing 2 micrograms per milliliter of brexpiprazole and 20 micrograms per milliliter of escitalopram oxalate, the assay results were striking: 99.67 percent recovery for brexpiprazole with a relative standard deviation of 0.308 percent, and 99.94 percent for escitalopram oxalate with a relative standard deviation of 0.227 percent. These figures indicate that the procedure measures the actual content of a mixture with near-perfect fidelity and minimal scatter, exactly the profile a quality control laboratory needs when certifying batches of a finished product.

What distinguishes this study from a routine validation exercise is its systematic greenness assessment. The authors evaluated the method with three complementary tools drawn from Green Analytical Chemistry. The AGREE metric, which scores a procedure against the twelve principles of green analytical chemistry, returned 0.71, indicating good compliance. The Blue Applicability Grade Index, which weighs practical applicability alongside sustainability, scored 72.5, and the Complex GAPI pictogram, which accounts for reagent hazards, energy consumption, and waste generation across the whole procedure, scored 81. The environmental arithmetic is straightforward: each ten-minute analysis consumes roughly ten milliliters of mobile phase, split into 5.5 milliliters of methanol and 4.5 milliliters of water, and generates only about ten milliliters of waste. The simple methanol-water system avoids more hazardous solvents entirely, and the short run time keeps energy demand low.

The team also extended the evaluation into White Analytical Chemistry, a framework that mirrors the twelve green principles but adds two further pillars: red for analytical performance and blue for practical and economic factors. By reporting the arithmetic means for the red, green, and blue sections separately, the authors acknowledged that a truly sustainable method must be green without sacrificing accuracy, precision, or cost-effectiveness. Their conclusion was that the method is acceptably green and suitable for sustainable routine applications without compromising performance, with only minor limitations remaining.

The practical significance of the work lies in its readiness for routine use. Because the method is the first created for rapid analysis of this binary mixture at a single wavelength, and because it passed every ICH Q2(R2) validation parameter with strong margins, pharmaceutical laboratories could adopt it directly for stability studies, formulation development, and batch release testing of any future brexpiprazole-escitalopram combination product. The combination of a ten-minute run time, modest solvent consumption, sub-2-percent variability, and recoveries near 100 percent represents a template for how modern analytical chemistry can serve drug development while shrinking its own environmental footprint, a balance that regulators and manufacturers alike are increasingly demanding.

Subject of Research: Development and greenness assessment of a validated RP-HPLC method for simultaneous estimation of brexpiprazole and escitalopram oxalate

Article Title: Development, validation, and greenness assessment of a green RP-HPLC method for rapid simultaneous estimation of brexpiprazole and escitalopram oxalate in combined dosage form

Article References: Solanki, D., & Dedania, Z. (2026). Development, validation, and greenness assessment of a green RP-HPLC method for rapid simultaneous estimation of brexpiprazole and escitalopram oxalate in combined dosage form. Discover Chemistry, 3(1), Article 444. https://doi.org/10.1007/s44371-026-00887-z

Image Credits: AI Generated

DOI: 10.1007/s44371-026-00887-z

Keywords: RP-HPLC, brexpiprazole, escitalopram oxalate, method validation, ICH Q2(R2), green analytical chemistry, AGREE metric, pharmaceutical analysis, liquid chromatography, antidepressants, quality control, sustainable chemistry