Why quantify aspirin and sildenafil together—and why salicylic acid matters
Co-administration of acetylsalicylic acid (ASA) and sildenafil citrate (SIC) is more than a pharmacological curiosity. The pairing has been explored for treating thin endometrium and for improving vascular erectile dysfunction—use cases in which cardiovascular risk and endothelial tone coincide. In these contexts, aspirin’s antiplatelet protection and sildenafil’s vasodilatory effect can be clinically complementary, a combination that invites fixed-dose co-formulation or, at the very least, joint quality control (QC) testing. The catch is obvious to anyone who has handled ASA: it hydrolyzes to salicylic acid (SA), and that degradant refuses to be ignored during analysis.
The analytical requirement follows directly from the pharmacology and chemistry. If a manufacturer releases a product containing aspirin and sildenafil, regulators and clinicians will expect proof that both actives are present at the right dose and that aspirin’s degradation is controlled. That proof must work on real samples and survive routine QC—for example, blends, finished dosage units, and stress conditions—without elaborate preparation or expensive consumables. UV spectrophotometry is attractive here because it is accessible, fast, and—if designed well—remarkably selective. The problem, historically, is selectivity in the face of heavy spectral overlap.
The paper at hand tackles exactly that problem and does so with three mathematically elegant UV methods designed to quantify ASA and SIC while explicitly accounting for SA in the same cuvette. The aim is not just “it works once” but “it works robustly, validates cleanly, and respects green analytical chemistry.” It is a refreshing shift from solvent-hungry chromatography to solvent-light UV, delivered with full validation and eco-metrics.
The analytical problem in one picture: overlapping spectra and what to do about them
Between 200 and 400 nm, the zero-order (D0) spectra of ASA, SIC, and SA sit on top of one another to a frustrating degree. ASA’s spectrum strongly overlaps with both SIC and SA across much of the range; after 300 nm, ASA fades, leaving SIC and SA to dominate. Direct UV measurement—pick a peak, draw a straight line—fails here because every “peak” belongs to more than one analyte. Spectral interference is not a nuisance; it is the central obstacle.
To resolve such mixtures without physical separation, modern UV methods exploit ratio spectra and carefully chosen wavelength arithmetic. By dividing a mixture spectrum by a standard spectrum of an interferent (“divisor”), one flattens the contribution of that interferent to a constant and frees the target analyte’s signature. Derivatives of ratio spectra can sharpen resolution further, and judicious two-wavelength differences can erase what remains of the interferer’s trace. The result is a family of techniques that trade solvent and time for a few lines of math—and when validated, they serve QC just as reliably.
In the ASA/SIC/SA system, three approaches—Dual Wavelength in Ratio Spectra (DW-RS), Advanced Amplitude Centering (AAC), and Double Divisor of Ratio-Difference Derivative (DDRD-D1)—were engineered to turn an intractable three-component overlap into three clean calibration functions. Each relies on a different way to neutralize the non-target spectra, but all share a nonnegotiable design principle: choose divisors and wavelengths where the non-targets behave predictably (equal amplitudes or straight baselines), so the measured signal depends only on the analyte you care about.
Three UV strategies that actually work (and when to pick which)
- DW-RS (Dual Wavelength in Ratio Spectra): Divide by a selected interferent’s spectrum, then use a pair of wavelengths where the second interferent has equal amplitudes; the difference in amplitudes (ΔP) reports the analyte concentration. For ASA and SIC, SA at 50 µg/mL served as divisor, with ΔP at 275.2–248.4 nm (ASA) and 253.7–275.2 nm (SIC). For SA, SIC at 40 µg/mL was the divisor, with ΔP at 308.0–325.0 nm.
- AAC (Advanced Amplitude Centering): Use a single divisor (again SA at 50 µg/mL), compute ΔP at selected wavelength pairs to obtain a “postulated” amplitude of each analyte at a centering wavelength (275.2 nm), subtract the constant of the divisor, and then read concentrations from a single-wavelength calibration. It is elegant and sensitive precisely because it collapses to one wavelength per analyte.
- DDRD-D1 (Double Divisor of Ratio-Difference Derivative): Build a double divisor from the sum of the two interferents’ spectra, form ratio spectra, take the first derivative (D1), and measure the analyte at a selective wavelength (ASA 262.0 nm, SIC 263.4 nm, SA 321.2 nm). The derivative step boosts selectivity and signal-to-noise for tightly overlapped regions.
The practical differences matter. DW-RS is simple and relies on intuitive “equal amplitude” conditions; it is transparent and easy to troubleshoot if a wavelength drifts. However, any error in selecting those paired wavelengths can generate “pseudo” results, so laboratories must verify wavelength positions and instrument calibration regularly. AAC improves sensitivity by collapsing the readout to a single wavelength after centering; because it uses a single divisor, it also streamlines routine work. DDRD-D1 often wins on selectivity when overlap is severe, at the cost of computing a derivative and preparing double divisors—still trivial with modern software but a step more than the others.
Wavelength choices are not decorative. They anchor the math to physical behavior—equal amplitudes for interferents or a straight-line region where a component contributes a constant. The study anchored ASA quantification at ΔP 275.2–248.4 nm (with SA as divisor) because SIC shows the same amplitudes at those points, cancelling itself out; SIC was anchored analogously at 253.7–275.2 nm; SA was read far from ASA’s influence, at 308.0–325.0 nm, since ASA does not absorb beyond ~300 nm. DDRD-D1 then selected derivative wavelengths that maximize selectivity: 262.0 nm (ASA), 263.4 nm (SIC), and 321.2 nm (SA). This disciplined wavelength logic is why the three methods agree so well.
How the methods were built and validated—what the data say
All UV work was performed on a JASCO V-650 using 1 cm quartz cells and 200–400 nm scans. Ethanol—analytical grade—was the sole solvent for standards and samples. Stock standards were 500 µg/mL; working solutions were 50 µg/mL for each analyte. Linearity ranges covered real-world formulations: 20–100 µg/mL for ASA, 5–50 µg/mL for SIC, and 4–60 µg/mL for SA. Across methods, coefficients of determination were ~0.999–0.9999, indicating near-ideal linear behavior within the validated windows.
Accuracy and precision were demonstrated in the usual ways—pure standards at three levels (e.g., ASA 30/50/70 µg/mL) and standard-addition on pharmaceutical preparations—returning mean recoveries close to 100% with repeatability and intermediate precision RSD < 2%. Importantly, laboratory synthetic mixtures were prepared at diverse ASA:SIC:SA ratios to stress the math. The methods sustained recovery near 98–103% across these blends, reinforcing selectivity in the presence of deliberately varied interferent levels.
Real-tablet application is the litmus test. Because an ASA/SIC co-formulated product was not commercially available, the team simulated a combination using marketed single-ingredient tablets (Aspirin® 100 mg; Vega® 50 mg base equivalent). Powder equivalent to 75 mg ASA and 25 mg SIC was extracted into ethanol, sonicated for 8 minutes, filtered, and diluted to nominal 90 µg/mL ASA and 30 µg/mL SIC for quantification. All three UV methods measured the actives in these “suggested formulations,” and the results were statistically indistinguishable from a proclaimed HPLC reference method based on t-tests, F-tests, and one-way ANOVA (no significant differences at p < 0.05). In other words: chromatographic rigor, spectrophotometric footprint.
Sustainability is not a slogan: putting greenness metrics to work
Moving beyond “uses less solvent,” the study applied four complementary green metrics. First, the Green Solvents Selecting Tool (GSST) framed solvent choice with a composite score G=H×S×E×W4G=\sqrt[4]{H \times S \times E \times W}G=4H×S×E×W across Health, Safety, Environment, and Waste categories (1–10 scale). Ethanol (used for UV) scored 6.6 (W 4.2, H 8.9, E 6.7, S 7), while acetonitrile (used in the reference HPLC) scored 5.8 (W 2.8, H 5.9, E 8.9, S 7). The implication for QC is straightforward: if UV gives equivalent results, ethanol is the greener default.
Second, the Greenness Index with spider diagram visualized Safety-Health-Environment attributes from safety data sheets. Ethanol’s spider chart occupied the “safe side,” whereas acetonitrile (and to a lesser extent the HPLC buffer component KH₂PO₄) pulled the HPLC profile toward lower-safety quadrants. Here the nuance matters: KH₂PO₄ is not particularly hazardous; acetonitrile is the main driver of risk and waste in the chromatographic workflow.
Third, the AGREE tool—the 12-principle Green Analytical Chemistry pictogram—returned 0.84 for the UV methods versus 0.71 for the HPLC procedure. That global score reflects minimal sample preparation, low solvent consumption, and straightforward instrumentation. Fourth, the Complex GAPI pictogram mapped impacts from sampling to waste. For UV, waste per analysis was <10 mL ethanol with an E-factor <0.1, and no waste treatment was needed; for HPLC, multiple reagent steps, higher temperature (~40 °C), and larger volumes of toxic organic solvent increased both impact and E-factor. These orthogonal metrics point in the same direction: UV achieves analytical goals with materially lower environmental cost.
If you prefer your take-home as a checklist rather than a manifesto, here is the pragmatic view of these tools:
- GSST helps you choose solvents before you start; AGREE and Complex GAPI help you judge the overall method afterward; the spider diagram helps you explain the trade-offs to non-chemists with a picture that maps risk, odor, fire safety, stability, and general characteristics at a glance.
A practical playbook for quality-control laboratories
Begin with solvent and standards. If your lab already stocks ethanol, you have everything you need for standards and samples; prepare 500 µg/mL stocks and 50 µg/mL working solutions for ASA, SIC, and SA. Keep a fresh SA stock on hand—ironic, yes, but measuring aspirin’s degradant accurately demands the degradant itself be well characterized. Calibrate in the validated ranges (ASA 20–100 µg/mL; SIC 5–50 µg/mL; SA 4–60 µg/mL), and verify linearity and residuals upon instrument qualification or after lamp replacement.
Choose the method like you choose a wrench: by fit. If your spectra are stable and your instrument’s wavelength accuracy is well controlled, AAC offers the simplest routine—single divisor, single readout wavelength (275.2 nm) after centering, and high sensitivity. If you prefer maximal transparency and diagnostic control, DW-RS lets you see exactly how “equal amplitude” conditions cancel interferents; it is ideal for training analysts because each subtraction is physically motivated. When you expect the heaviest overlap or modest baseline curvature, DDRD-D1 buys selectivity by derivative processing and double divisors—excellent when matrix components threaten to sneak under the tent. Document your rationale; regulators appreciate method-to-purpose alignment.
For sample preparation, the study’s tablet protocol is a good template: weigh, extract into ethanol, sonicate 8 minutes, filter, and dilute to target concentrations (e.g., 90 µg/mL ASA and 30 µg/mL SIC for the combined assay). Build a small system-suitability set: blank ethanol; divisor spectrum(s) at the validated concentration(s); a midpoint standard; and a check solution containing SA at a realistic degradation level (for stressed lots). Add bracketing standards every 10 samples; apply standard-addition on at least one finished product lot to confirm matrix tolerance. Keep an eye on ΔP positions (e.g., 275.2–248.4 nm for ASA in DW-RS): a 0.2 nm drift is usually benign, but a 2 nm shift is a red flag—re-qualify or re-optimize.
Limitations, caveats, and sensible extensions
No spectrophotometric method is invincible. These approaches assume the only UV-active species in the selected windows are ASA, SIC, and SA; excipients with significant absorbance near 250–330 nm could disturb the equal-amplitude or straight-line assumptions. The validation used marketed tablets and synthetic mixtures but not, for example, syrup matrices or complex excipient systems; such products might need matrix-matched divisors or additional clean-up. Fortunately, the mathematics is flexible: if an excipient intrudes at 275.2 nm, alternative equal-amplitude pairs can be selected with fresh scans and the same logic.
Ethanol as a universal solvent is a strength for greenness, but not every formulation dissolves identically in ethanol. If dissolution is slow or incomplete, a small co-solvent fraction may be tempting. Resist the urge to “just add acetonitrile” unless you have re-established linearity and recalculated greenness; the environmental advantage can erode quickly. A better first step is to optimize sonication time, temperature (without approaching aspirin’s hydrolysis sweet spot), and filtration. Keep extraction gentle and brief; the goal is reproducible solubilization, not stress testing.
Finally, while equivalence to HPLC was statistically demonstrated in this study, your own method transfer will require due diligence. Confirm accuracy and precision in your hands, under your SOPs, with your lamps and cells. If your regulatory filing names a chromatographic assay, these UV methods are best positioned as validated alternatives for routine release and in-process control, with chromatography retained for investigations or where compendial obligations require it. The upside is not theoretical: fewer toxic solvents, faster cycle time, and lower cost per test—sustainable both environmentally and operationally.
Bottom line
Three mathematically sound, experimentally validated, and eco-conscious UV methods—DW-RS, AAC, and DDRD-D1—can simultaneously quantify aspirin and sildenafil while controlling for aspirin’s degradant, salicylic acid, in routine QC. They deliver linearity across practical ranges, accuracy and precision within RSD < 2%, and statistical parity with an HPLC reference, all while replacing acetonitrile-heavy workflows with ethanol-based spectroscopy. When you can get HPLC-grade answers with a few carefully chosen wavelengths and greener solvent, the smart choice—pun fully intended—is clear.
FAQ
1) Can these UV methods replace HPLC in a regulated environment?
They matched an HPLC reference statistically (no significant differences by t-test, F-test, and one-way ANOVA at p < 0.05) in simulated co-formulations and synthetic mixtures. In practice, many labs run them as validated routine assays or IPCs and retain HPLC for investigations or where compendial methods dictate. Plan a formal method transfer to document equivalence in your own facility.
2) When is AAC preferable to DW-RS or DDRD-D1?
If your instrument’s wavelength accuracy is stable and your matrix is simple, AAC offers high sensitivity with a single divisor and a single readout wavelength after centering—fast to run, simple to train, and robust day-to-day. Choose DDRD-D1 when overlap is severe or baseline curvature is a concern; choose DW-RS when you want maximum transparency and easy diagnostic checks on the equal-amplitude conditions.
3) What if my aspirin is highly hydrolyzed—does excess SA break the methods?
The methods were engineered to account for SA explicitly, not to wish it away. Both DW-RS and AAC use SA as a divisor, and DDRD-D1 includes SA in the double divisor; validation across multiple ASA:SIC:SA ratios maintained recoveries near 98–103%. If SA levels exceed your calibration design, extend the SA linear range and reconfirm wavelength conditions; the mathematics remains valid as long as you recalibrate.
Source: All data, wavelengths, validation ranges, greenness scores, and procedures summarized here are drawn from the Heliyon 2023 research article on UV strategies for concurrent quantification of aspirin and sildenafil citrate in the presence of salicylic acid, including AGREE, Complex GAPI, GSST, and spider-diagram assessments.
