Greener, Faster HPLC Test Puts Two Prostate Drugs Under One Precise Lens

When India’s drug regulator approved a fixed-dose capsule combining silodosin and tadalafil for enlarged prostate in October 2023, quality-control laboratories gained a new problem: how to measure two very different molecules, plus every fragment they shed as they break down, in a single reliable run. A team at Laxminarayan Dev College of Pharmacy in Bharuch, Gujarat, now reports a solution that is not only accurate and sensitive but also explicitly scored for its environmental footprint. Writing in Discover Chemistry, Yukti Rana and colleagues describe a stability-indicating reverse-phase high-performance liquid chromatography method that quantifies both drugs simultaneously in a synthetic mixture, validates every parameter demanded by the ICH Q2(R2) guideline, and then goes a step further by pitting the method against a published competitor using one-way analysis of variance.

The clinical context matters. Benign prostatic hyperplasia, the non-cancerous enlargement of the prostate that plagues aging men, produces a familiar cluster of miseries: weak urinary stream, nocturia, hesitancy, and incomplete bladder emptying. Silodosin attacks the problem as a highly selective alpha-1A adrenoceptor antagonist, relaxing smooth muscle in the prostate and urethra so urine flows more freely. Tadalafil works through a different route, raising cyclic guanosine monophosphate signaling that is thought to improve tissue oxygenation, dampen inflammation, and curb cell proliferation in prostatic tissue, while its vasodilatory action also supports erectile function. Together the two drugs offer dual benefit, which is precisely why regulators allowed them into one capsule and why analytical chemists needed one method that could certify both at once.

The chromatographic recipe is deliberately economical. Separation occurs on an Inertsil C18 column, 250 by 4.6 millimeters with five-micron particles, run in isocratic mode with a mobile phase of 0.02 molar phosphate buffer at pH 5 mixed with methanol in a 34:66 volume ratio. The flow rate is a modest 1.0 milliliter per minute, detection sits at 220 nanometers where both drugs absorb strongly, and the column temperature is held at 20 degrees Celsius. Under these conditions silodosin elutes at roughly 3.810 minutes and tadalafil at 10.165 minutes, a clean temporal gap that leaves ample room for degradation products without the gradient pumping and solvent switching that inflate cost and waste. The authors used a Shimadzu LC-2010 CHT system with UV detection, and working standards supplied by Sun Pharmaceutical came in at purities above 99.5 percent.

Validation numbers are the currency of analytical chemistry, and this method pays out consistently. Calibration was linear from 40 to 120 micrograms per milliliter for silodosin and 25 to 75 micrograms per milliliter for tadalafil, with correlation coefficients of 0.9983 for both compounds. Recovery studies at three spiking levels returned values between 99.34 and 100.98 percent for silodosin and 98.51 to 99.78 percent for tadalafil, showing that common capsule excipients such as mannitol, microcrystalline cellulose, and magnesium stearate do not interfere. Repeatability produced relative standard deviations of 0.4377 percent for silodosin and 0.4998 percent for tadalafil, and every intraday, interday, and robustness test stayed below the two percent RSD threshold that regulators treat as the boundary of acceptable variability.

Sensitivity reaches into territory that matters for trace impurity work. Using the standard deviation of intercepts and calibration slopes as prescribed by ICH Q2(R2), the team calculated detection limits of 0.002686 micrograms per milliliter for silodosin and 0.002338 micrograms per milliliter for tadalafil, with quantification limits of 0.008139 and 0.007085 micrograms per milliliter respectively. Those nanogram-scale thresholds mean the method can spot the earliest chemical whispers of decomposition long before they approach pharmacopoeial action levels. System suitability testing with six replicate injections confirmed adequate theoretical plates, tailing factors, and resolution, and deliberate perturbations of mobile phase composition, flow rate, pH, and temperature left the results essentially unmoved.

The stability-indicating claim rests on forced degradation, the practice of deliberately breaking drugs apart to prove a method can still find the survivors. The researchers exposed the synthetic mixture to one normal hydrochloric acid and sodium hydroxide for six hours, three percent hydrogen peroxide, direct sunlight for two days, and a 60 degree Celsius hot air oven for 24 hours. Silodosin degraded most under oxidative stress and least under heat, while tadalafil was most vulnerable to acid and most heat-stable; neither drug showed significant breakdown under basic or photolytic conditions. Crucially, every degradant peak resolved from the parent peaks with resolution values above 2.0, meaning the assay can certify how much intact drug remains even in a chemically battered sample.

What distinguishes this study from the long line of HPLC validations is its environmental accounting. The team ran four complementary greenness tools. The Green Analytical Procedure Index painted a mostly green-and-yellow pentagram with a single red segment for waste treatment, marking the method as moderately green. The AGREE metric, scored against the twelve principles of green analytical chemistry, returned 0.75 on a zero-to-one scale, while AGREEprep, which judges only the sample preparation, scored 0.69. The Blue Applicability Grade Index, which weighs practical suitability alongside sustainability, landed at 77.5. Methanol-buffer systems carry a moderate solvent burden, the authors concede, but the scores sit comfortably alongside recent eco-friendly HPTLC and quality-by-design HPLC methods reported for carvedilol, amlodipine-indapamide, and teneligliptin-pioglitazone combinations.

The statistical showdown adds a layer of rigor that method-development papers rarely attempt. Comparing assay values from the new method against a previously published RP-HPLC method from the Research Journal of Pharmacy and Technology, one-way ANOVA found highly significant differences for tadalafil (F = 144.40, p = 0.00027) and significant differences for silodosin (F = 17.04, p = 0.01452). Significance here is not a flaw but the point: the new method’s higher mean assay values trace to lower variability, sharper peak symmetry, reduced baseline noise, dramatically lower detection limits, and, most tellingly, demonstrated separation of degradation peaks, a capability the older method never showed. A separate ANOVA across the five stress conditions found no significant differences (p > 0.05), confirming the method reads consistently whether the sample is pristine or degraded.

For the pharmaceutical industry the practical implications are straightforward. A single isocratic run that quantifies both actives, shrugs off excipient interference, survives deliberate abuse of its parameters, and resolves every degradant is exactly what routine quality control and regulatory stability submissions require, and doing it with a phosphate buffer and methanol rather than acetonitrile-heavy gradients keeps solvent budgets and hazardous waste comparatively lean. The authors note the method is ready for application to combined dosage forms and synthetic mixtures, and its full ICH Q2(R2) compliance package should ease its path into pharmacopoeial workflows. As green analytical chemistry matures from slogan to scoring system, this study offers a template: validate the numbers, grade the footprint, and then let statistics referee the contest against the incumbents.

Subject of Research: Green stability indicating RP- HPLC method for simultaneous estimation of Silodosin and Tadalafil development validation and comparative evaluation using one way ANOVA

Article Title: Green stability indicating RP- HPLC method for simultaneous estimation of Silodosin and Tadalafil development validation and comparative evaluation using one way ANOVA

Article References: Rana, Y., Mandale, D. D., Ugharatdar, D. S., & Chauhan, D. N. (2026). Green stability indicating RP- HPLC method for simultaneous estimation of Silodosin and Tadalafil development validation and comparative evaluation using one way ANOVA. Discover Chemistry, 3(1), Article 452. https://doi.org/10.1007/s44371-026-00849-5

Image Credits: AI Generated

DOI: 10.1007/s44371-026-00849-5

Keywords: Green, stability, indicating, HPLC, method, simultaneous, estimation, Silodosin, Tadalafil, development, validation, comparative