Considerations Regarding the Use of Fresh Urine in Microplastic and Nanoplastic Analysis vs. Urine Samples That Have Undergone Complex Processing

Abstract

Microplastic and nanoplastic (MNP) detection in human urine has emerged as an area of increasing scientific and public interest. However, reported urinary MNP concentrations vary substantially across the literature due to differences in collection methods, storage conditions, sample digestion protocols, filtration workflows, and analytical instrumentation. Most published urine MNP studies rely on delayed and highly processed laboratory workflows involving refrigeration or freezing, alkaline digestion, filtration, centrifugation, and spectroscopic analysis.

These workflows may alter the native biological matrix and introduce variability related to sample aging, oxidation, pH changes, bacterial growth, particle loss, and polymer surface modification.

This technical note discusses considerations regarding the use of fresh urine for real-time analysis compared with urine specimens that have undergone prolonged storage and complex laboratory preprocessing. Particular attention is given to matrix fidelity, the potential effects of delayed processing, and the limitations of conventional methods in detecting nanoplastics. The note also discusses the conceptual framework of decentralized, real-time intact-liquid analysis using the EcoExposure™ platform. Importantly, aged urine experiments described herein were utilized as extreme stress-test conditions rather than intended workflow conditions. Fresh or near-fresh urine remains the preferred and intended sample condition for preserving native matrix characteristics and minimizing artifact introduction.

This paper is also available at: Abstract

Microplastic and nanoplastic (MNP) detection in human urine has emerged as an area of increasing scientific and public interest. However, reported urinary MNP concentrations vary substantially across the literature due to differences in collection methods, storage conditions, sample digestion protocols, filtration workflows, and analytical instrumentation. Most published urine MNP studies rely on delayed and highly processed laboratory workflows involving refrigeration or freezing, alkaline digestion, filtration, centrifugation, and spectroscopic analysis.

These workflows may alter the native biological matrix and introduce variability related to sample aging, oxidation, pH changes, bacterial growth, particle loss, and polymer surface modification.

This technical note discusses considerations regarding the use of fresh urine for real-time analysis compared with urine specimens that have undergone prolonged storage and complex laboratory preprocessing. Particular attention is given to matrix fidelity, the potential effects of delayed processing, and the limitations of conventional methods in detecting nanoplastics. The note also discusses the conceptual framework of decentralized, real-time intact-liquid analysis using the EcoExposure™ platform. Importantly, aged urine experiments described herein were utilized as extreme stress-test conditions rather than intended workflow conditions. Fresh or near-fresh urine remains the preferred and intended sample condition for preserving native matrix characteristics and minimizing artifact introduction.

This paper is also available at: https://doi.org/10.5281/zenodo.20378899

Introduction

Human exposure to microplastics and nanoplastics has become an area of growing concern due to the increasing presence of synthetic particulate materials in water, food, air, and consumer products. In recent years, investigators have reported the detection of microplastics in multiple human biological matrices, including blood, placenta, semen, breast milk, tissue samples, and urine.

Urine has emerged as a particularly attractive matrix for investigation because it is non-invasive, widely accessible, and compatible with decentralized collection. However, substantial heterogeneity exists across published urinary MNP studies. Reported concentrations differ by multiple orders of magnitude, and analytical methods vary considerably between research groups.

Most published approaches rely on multi-step laboratory processing workflows that involve prolonged storage and destructive preprocessing prior to analysis. Common workflows include refrigeration or freezing, alkaline digestion using potassium hydroxide (KOH), proteinase digestion, filtration, centrifugation, and spectroscopic analysis using microRaman, μFTIR, pyrolysis GC/MS, or related techniques.

While these laboratory methods have advanced the field substantially, relatively little discussion has focused on how sample aging and extensive preprocessing may alter the biological matrix prior to analysis. These considerations may be particularly relevant in urine, which undergoes known chemical and biological changes following collection.

2. Freshness and Matrix Fidelity in Urine Testing

In both clinical chemistry and consumer wellness testing, fresh or near-fresh urine specimens are commonly preferred to preserve matrix integrity and minimize artifacts associated with prolonged storage. Delayed urine processing is known to alter multiple physical, chemical, and biological characteristics of the sample.

These changes may include: oxidation of urinary compounds, pH drift, ammonia generation, bacterial proliferation, crystal formation, turbidity changes, mucus precipitation, cellular degradation, and alterations in optical appearance.

Such changes are the reason many clinical urine analyses and over-the-counter wellness tests emphasize prompt analysis or standardized storage conditions.

Table 1. Representative Changes Observed in Fresh Versus Aged Urine

Component / Feature	Fresh Urine	Aged / Delayed Urine	Potential Effect
Color	Pale yellow / clear	Darkened / oxidized	Altered optical properties
pH	Typically mildly acidic	Often becomes alkaline	Matrix chemistry changes
Ammonia	Low	Increased	Protein and bacterial effects
Bacteria	Minimal	Increased proliferation	Biological variability
Turbidity	Lower	Increased cloudiness	Optical interference
Crystals	Variable	May increase	Particle-like artifacts
Cellular Integrity	Preserved	Cellular degradation	Matrix instability
Mucus / Protein Threads	Minimal	Often more visible	Heterogeneous optical behavior

These factors highlight the importance of considering matrix fidelity when interpreting urinary MNP measurements.

Figure 1. Representative Appearance of Fresh Versus Aged Urine Samples. Visual comparison of fresh urine (right) and aged urine (left) demonstrating qualitative matrix changes that may occur during delayed storage. The aged sample demonstrates darker coloration and increased visual heterogeneity consistent with known oxidative, chemical, and biological changes that occur in urine over time, including oxidation of urinary compounds, pH shifts, and increased turbidity. These observations highlight the potential importance of sample freshness and matrix fidelity in urinary microplastic and nanoplastic analysis workflows.

3. Limitations of Conventional Laboratory Processing Approaches

A common assumption in analytical science is that highly processed laboratory workflows are inherently more precise and reliable than decentralized or minimally processed approaches. However, in the context of urinary MNP analysis, prolonged and complex processing may itself introduce additional variability.

Most published urinary MNP studies utilize workflows involving:

• delayed sample analysis,

• refrigeration or freezing,

• alkaline digestion,

• filtration,

• centrifugation,

• concentration steps,

• and extensive spectroscopic preparation.

Potassium hydroxide (KOH), commonly used in MNP studies, is not intended to digest plastic particles directly. Rather, it is used to digest biological material surrounding the particles.

However, prolonged alkaline exposure may contribute to polymer surface weathering, oxidation, fragmentation, or altered spectroscopic signatures in certain materials.

Similarly, filtration and centrifugation workflows may introduce particle loss, particularly for very small particles or true nanoplastics. Multiple transfer and washing steps may further contribute to variability between laboratories.

Additionally, many currently utilized spectroscopic techniques possess practical lower size detection limits in the micrometer range and may not reliably characterize true nanoplastics below those thresholds.

These methodological considerations may contribute to some of the substantial heterogeneity observed across published urinary MNP studies.

Figure 2. Fresh Real-Time Analysis vs. Delayed Complex Processing in Urinary Microplastic and Nanoplastic Workflows. Conceptual comparison between the EcoExposure™ real-time intact-liquid urine analysis workflow (left) and conventional laboratory-based urinary microplastic and nanoplastic processing workflows (right). The EcoExposure™ approach emphasizes fresh urine analysis, minimal preprocessing, decentralized smartphone-compatible imaging, and rapid temporal optical assessment within approximately 30–60 minutes. In contrast, many conventional laboratory workflows involve refrigeration or freezing, transport, chemical digestion, filtration, centrifugation, spectroscopic preparation, and delayed Raman, FTIR, or Py-GC/MS analysis over periods ranging from hours to days. The figure is intended as a high-level conceptual comparison and does not depict proprietary assay chemistry or analytical algorithms.

4. Real-Time Intact Liquid Testing and Decentralized Urine Analysis

The EcoExposure™ platform was conceptually developed around the principle of real-time intact-liquid analysis using minimal sample preprocessing. Rather than relying on prolonged destructive workflows, the platform evaluates temporal optical interaction behavior directly within the liquid matrix.

The broader concept emphasizes:

• fresh or near-fresh sampling,

• intact liquid analysis,

• minimal processing,

• decentralized usability,

• smartphone compatibility,

• and rapid temporal assessment.

Importantly, this technical note does not disclose proprietary reagent formulations, analytical algorithms, calibration workflows, or computational methodologies associated with the EcoExposure™ platform.

The purpose of this discussion is instead to highlight broader methodological considerations regarding sample freshness and matrix preservation in urinary MNP analysis.

5. Existing Consumer and Wellness Urine Testing Paradigms

The concept of decentralized urine testing is already widely accepted across healthcare and wellness applications. Numerous consumer and wellness products emphasize rapid or near-real-time analysis using minimally processed fresh urine samples.

Table 2. Examples of Existing Consumer and Wellness Urine Testing Approaches

Product / Category	Intended Use	Smartphone / App Integration	Fresh Sample Use
Vivoo	Wellness / hydration	Yes	Yes
Oova	Fertility / hormone tracking	Yes	Yes
Pregnancy Tests	hCG detection	Sometimes	Yes
Ketone Test Strips	Metabolic monitoring	Sometimes	Yes
UTI Test Strips	Leukocyte / nitrite screening	Sometimes	Yes

These existing paradigms demonstrate that decentralized fresh urine analysis is already a broadly established concept in multiple healthcare and wellness settings.

6. Aged Urine as an Extreme Stress-Test Condition

In exploratory EcoExposure™ experiments, aged urine specimens were intentionally evaluated as extreme stress-test conditions. These samples demonstrated expected visual and chemical changes associated with delayed storage, including color shifts, increased turbidity, and altered matrix behavior.

Notably, detectable optical interaction behavior remained observable even under these degraded conditions. However, these experiments were not intended to represent the preferred workflow for analysis.

Rather, the observations suggest that assay functionality may remain partially observable despite substantial matrix degradation and storage-related changes.

Fresh or near-fresh urine remains the intended and preferred sample condition for preserving biological fidelity and minimizing artifacts associated with prolonged storage and extensive laboratory preprocessing.

7. Timing Considerations in Real-Time Versus Conventional Workflows

One conceptual distinction between real-time intact-liquid workflows and many current laboratory approaches is total elapsed time between specimen collection and analysis.

Conventional urinary MNP workflows may involve:

1. sample collection,

2. transport,

3. refrigeration or freezing,

4. digestion,

5. filtration,

6. centrifugation,

7. drying,

8. spectroscopic preparation,

9. and eventual analytical characterization.

These workflows may span many hours to multiple days depending on protocol complexity.

In contrast, decentralized real-time workflows conceptually aim to evaluate the sample closer to its native post-collection state.

A representative figure comparing these timelines is included in this technical note.

8. Discussion

As interest in urinary MNP analysis continues to expand, methodological standardization will become increasingly important. Current literature demonstrates substantial heterogeneity in reported urinary MNP concentrations and analytical workflows.

While advanced laboratory instrumentation remains critically important for confirmatory analysis and polymer characterization, considerations regarding freshness, matrix preservation, and preprocessing artifacts may warrant greater discussion within the field.

The observations discussed in this technical note suggest that:

• delayed processing may alter urine matrix characteristics,

• destructive preprocessing may introduce variability,

• filtration-based workflows may incompletely capture nanoplastics,

• and fresh intact-liquid analysis may preserve additional biologically relevant information.

Further investigation will be necessary to determine how these variables influence downstream analytical interpretation and biological relevance.

Conclusion

The rapidly evolving field of urinary microplastic and nanoplastic analysis presents substantial opportunities as well as methodological challenges. While conventional laboratory workflows remain foundational to current research, considerations regarding freshness, matrix fidelity, and extensive preprocessing deserve additional attention.

Fresh or near-fresh urine analysis may preserve native biological characteristics that are altered during delayed and destructive workflows. These considerations may become increasingly important as investigators attempt to characterize smaller particulate populations, including true nanoplastics, within complex biological matrices.

The broader concept of decentralized real-time urine analysis, already well established across multiple wellness and healthcare applications, may provide a useful complementary framework for future urinary MNP investigations.