Introduction

Chronic mucoobstructive lung diseases, such as chronic obstructive pulmonary disease (COPD), asthma, Cystic Fibrosis (CF), and non-CF bronchiectasis, are characterized by mucus hyperconcentration1,2. They significantly burden the healthcare system worldwide, affecting an estimated 20–25% of the population. Despite their shared features, these diseases result in diverse clinical outcomes. Current diagnostic or prognostic approaches, including etiological factors, symptoms, spirometry, and lung imaging, lack a unified and objective test for diagnosis and prognosis. For instance, presently, chronic bronchitis (CB), component of COPD, diagnosis depends on subjective measures like the St George’s Respiratory Questionnaire (SGRQ) and American Thoracic Society (ATS) Questionnaire3, underscoring the need for objective biological markers for diagnosis and prognosis and encouraging patients and at-risk populations to acknowledge the severity of their condition.

Gel forming mucins, the principal glycoproteins in mucus responsible for its viscoelastic properties, have emerged as potential biomarkers for CB diagnosis, prognosis, exacerbation risk assessment, and monitoring therapies3,4. Therefore, accurate and sensitive measurement of mucin in clinical samples is essential in research and clinical applications.

Several analytical techniques have been used to detect and quantify the major lung mucins, MUC5AC and MUC5B individually, such as enzyme-linked immunosorbent assay (ELISA)5, agarose gel electrophoresis (AGE)6,7, and LC–MS-MS4,8,9. Both ELISA and AGE rely on antibody-based detection, which can introduce variability due to epitope availability (proteolysis) and masking and limited dynamic range. LC-MS-MS provides state-of-the-art, highly sensitive and accurate quantitation of MUC5AC and MUC5B and requires expensive instrumentation, well-defined isotopically labelled standards and rigorous sample preparation.

We have developed a robust, quantitative approach for total mucin concentration measurement that enables biophysical and absolute quantification of total mucins, regardless of mucin type using Size Exclusion Chromatography coupled with Multi-Angle Laser Light Scattering and Differential Refractometer [SEC-(MALLS)-dRI]3. In this technique, no standards and antibodies are required; large mucin macromolecules are separated with size-exclusion chromatography, and an inline dRI detector measures changes in the refractive index as the mucins elute, altering the solution’s optical properties. This change causes a measurable deflection of light in the sample cell relative to a reference cell containing only the pure solvent. The deflection is detected and converted into an electrical signal proportional to the solute concentration based on first principles of light refraction.

We previously used this technique in multiple clinical and research studies3,10,11,12,13. Aiming for broader clinical/healthcare setting use and research applicability, our study here introduces the detailed validation of total mucin measurement in induced or spontaneous sputum samples, offering high sensitivity, selectivity, and ease of use. This method, proposed as a reliable biomarker, could help the diagnosis, prognosis, and treatment monitoring of mucoobstructive airway diseases, ultimately helping to reduce morbidity and mortality.

Results

A typical SEC-(MALLS)-dRI chromatogram of the samples is shown in Supplementary Fig. 1. The differential refractive index (dRI) signal was used for quantitation, while the light scattering signal (detector #11, 90° angle) was used to confirm the elution of high molecular weight mucins, which is optional and not essential for this measurement.

Precision

Figure 1 compares the precision of SEC-(MALLS)-dRI measurements such as within run, between days, and within-lab precisions. The within-run precision of the measurement was evaluated and categorized into three levels: high, medium, and low. The high precision level exhibited a mean value of 2845.75 µg/ml ± 69.57 SD, and a coefficient of variation (CV %) of 2.44. The medium precision level had a mean value of 1433.94 µg/ml ± 14.57 SD, and CV % of 1.02. Lastly, the low precision level demonstrated a mean value of 465.56 µg/ml ± 12.52 SD with a CV % 2.69. These findings indicate excellent precision with low degrees of variability within each precision level between 1.02 and 2.69 (Fig. 1A, Supplementary Table 1).

Fig. 1
figure 1

Total mucin precision assays: (A) Within-run (intraassay) precision. The graph demonstrates excellent precision across low (%CV 2.4), medium (%CV 1.02), and high (%CV 2.69) mucin concentrations. (B) Between days (inter-assay) precision results indicate excellent reproducibility with SD: 8.12 and %CV 3.32. (C) Within-lab (intermediate) precision: The difference (%) in total mucin levels measured using two independent SEC–MALLS-dRI systems with manual (SD;11.13, % CV:3.83) and autoinjector (SD: 4.4, %CV;1.48) demonstrating high consistency with 2.11% precision.

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Between-day precision exhibited a mean value of 349.25 µg/ml ± 8.12 SD with 2.32 CV % (Fig. 1B, Supplementary Table 2). Precision was also tested for two different injection methods, auto injection vs manual injection, and the mean, SD, and CV % were calculated for seven replicates (Fig. 1C, Supplementary Table 3). The mean values for auto-injector and manual injection samples were 296,68 ± 4.4 SD, and 290,43 ± 11.1 SD respectively (P = 0.38). The CV %, which measures the relative variability in relation to the mean, was lower for the autoinjector group, 1.48%, compared to the manual injector group, 3.83%. Altogether, these data demonstrated excellent precision of the SEC-(MALLS)-dRI method for total mucin measurement.

Recovery and parallelism

Recovery studies were conducted to evaluate the method’s accuracy by spiking known concentrations of a mucin standard into sputum samples and calculating the percentage of the added mucin recovered during the analysis. The baseline mucin concentrations of the samples, prior to spiking with the standard, ranged from 542.83 µg/mL to 561.54 µg/mL, with a mean value of 552.35 µg/mL and a low coefficient of variation (CV%) of 1.69%, indicating consistent baseline measurements across replicates (Supplementary Table 4). The mean concentration of the added mucin was 75.28 µg/ml. After adding the mucin standard, the final concentrations measured were 616.54 µg/mL, 638.53 µg/mL, and 662.53 µg/mL, respectively. The mean final concentration was 639.20 µg/mL with a CV% of 3.5%, demonstrating high consistency in the post-spiking measurements. (Supplementary Table 4). The mean recovery was 116%, within the acceptable limit (+/− 20%), indicating that the method accurately detected and quantified the added mucins.

Parallelism is an important assessment to address relative accuracy by determining the effect of the dilution of the samples14. Parallelism was assessed to evaluate the accuracy and reliability of the SEC–(MALLS)-dRI method across a range of sample dilutions, ensuring that the assay response remains proportional to the analyte concentration. Two sputum samples were serially diluted across five levels and analyzed in duplicate. The measured mucin concentrations were compared against expected concentrations, and the percent recovery was calculated (Fig. 2A, Supplementary Table 5).

Fig. 2
figure 2

Parallelism and recovery study for total mucin concentrations. (A) Total mucin concentration across a serial dilution of two separate samples. (B) The graph shows the recovery of the serial dilutions. The % recovery was calculated using the formula: Observed Concentration/ Expected Concentration X 100. The dotted line represents + /− 20% deviation from the initial sample values (100%).

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The Percent Recovery was calculated. Acceptable recovery was required to be within 80–120% of the expected concentration (Fig. 2B, and Supplementary Table 5).

Specificity-selectivity and carry-over

Interference is the main factor determining the analytical specificity and selectivity of the method15. To evaluate specificity, sputum samples (N = 3) were spiked with potential contaminants, including saliva, blood, and DNA, to assess their impact on total mucin concentration measurements.

Minimal interference was observed at saliva concentrations ≤ 50%, with recovery remaining less than 3%, within acceptable limits (± 10% of the expected concentration). Even with 50% saliva contamination, the interference was modest, around 25%. (Fig. 3A, supplementary Fig. 2).

Fig. 3
figure 3

Specificity and interference assessment for total mucin quantification by SEC–MALLS-dRI: (A) Effect of saliva contamination on mucin quantification. Minimal interference was observed at saliva concentrations up to 20%, with recovery within acceptable limits. Significant deviations were noted at saliva concentrations of 50%. (B) Effect of blood contamination on mucin quantification. No significant interference was observed at blood concentrations ≤ 1%. However, interference increased progressively at concentrations ≥ 2%, exceeding acceptable limits (≥ 10%) at 2% and 5%. (C) Effect of DNA contamination and mitigation with DNase treatment. DNA concentrations ≤ 1% caused no interference, but significant deviations were observed at concentrations ≥ 2%. (D) DNase treatment effectively restored mucin measurement accuracy to within acceptable thresholds, eliminating interference. LSB = Light Scattering Buffer.

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No significant interference was observed at blood concentrations ≤ 1%, with recovery values remaining within acceptable limits (± 10% of the expected concentration). At higher blood concentrations (2% and above), however, substantial interference was detected, with deviations increasing progressively, exceeding the predefined acceptance threshold. At 10% blood concentration, interference exceeded 30% (Fig. 3B). These findings highlight that while minor blood contamination (≤ 1%) does not affect mucin quantification, higher blood levels introduce substantial errors, underscoring the need for proper sample collection protocols.

Similarly, no interference was observed at DNA concentrations ≤ 1%, with measured mucin concentrations aligning closely with true values. At DNA concentrations > 2% and 5%, deviations became significant, with recovery values exceeding acceptable limits. (Fig. 3C, supplementary Fig. 2). In parallel, DNase was applied to DNA-spiked samples to evaluate its impact on DNA-contaminated samples. The result demonstrated that accurate measurement of total mucin concentrations required DNase treatment at higher DNA contaminations (% 20) (Fig. 3D). DNase treatment, the interference caused by high DNA concentrations was effectively mitigated, restoring measurement accuracy to within acceptable thresholds.

These findings indicate that the SEC-(MALLS)-dRI method is highly selective but sensitive to DNA contamination. DNase treatment is essential for samples from conditions like CF, where extracellular DNA levels in sputum are significantly elevated due to increased neutrophilic inflammation and NETosis.

For the carry-over assessment, blank samples were injected after injecting two high-level sputum samples (Total mucin concentration: 3696.52 and 1327.46 µg/mL), but no detected peak was observed for calculation. As a result, no carry-over was present, and the system’s suitability was determined (Supplementary Table 6).

Linearity, analytical measurement range, LLOQ, ULOQ

Linearity was evaluated in two independent experiments conducted on different days and different concentration ranges. The first experiment assessed nine concentration levels, ~ 30 to ~ 4000 µg/ml (supplementary Fig. 3), while the second covered seven concentration levels, ~225–14,500 µg/ml (Fig. 4). Typically, the minimum reliable detection limit of the dRI detector is 1 µg per injection per mucin peak. Applying a 20-fold dilution factor corresponds to a detection limit of 20 μg/ml per sample. For the upper limit, the detector exhibits a broad dynamic range. It does not saturate easily, with the high-range reaching up to 1000 µg per injection per mucin peak without dilution.

Fig. 4
figure 4

Linearity and dynamic range of total mucin quantification by SEC–MALLS-dRI. (A, B) The lower limit of quantitation (LLOQ) was determined to be 29.19 µg/mL, and the upper limit of quantitation (ULOQ) was established at 14,525 µg/mL with our most concentrated sample. Multiple injection of low- and high- concentration samples within the analytical range demonstrated high precision. (C, D) The linear relationship between the expected (nominal) and observed mucin concentrations across a wide analytical range (~ 200–14,500 µg/mL) is shown. The assay demonstrates excellent linearity with an R2 value of 0.9974, confirming the method’s robustness and suitability for quantifying mucin concentrations across diverse clinical and research applications.

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Low and high samples were analyzed to establish LLOQ and ULOQ. The analytical range of the assay spanned from ~ 30 µg/mL to ~ 14,500 µg/ml. The lower limit of quantitation (LLOQ) was determined to be 29.19 µg/mL, and the upper limit of quantitation (ULOQ) was established at 14,525.00 µg/ml with our most concentrated sample. (Fig. 4A,B). The observed concentrations of total mucin were plotted against the expected concentrations. A linear correlation with an r-squared value of 0.9974 was observed for total mucin measurement (Fig. 4C,D).

Stability

The assessment of frozen (− 20 °C) mucus sample stability showed that the clinical samples remained stable for 10 months, with results within 20% of the nominal levels. (Fig. 5A, supplementary table 7). Freeze–thaw stability (2 cycles) also yielded results at nominal retention of 10% compared with fresh samples (supplementary table 7).

Fig. 5
figure 5

Long-term stability of total mucin concentrations under frozen (naïve) and refrigerated (4MGuHCl) conditions. (A) Stability of mucins concentration in sputum samples stored frozen at − 20 °C in PBS for up to 16 months. Measurements were taken at baseline and monthly, with deviations from baseline consistently within ± 20% up to 14 months, demonstrating stability up to 14 months. (B) Stability of mucins concentration in sputum samples stored at 4 °C in 4 M GuHCl for up to 16 months. Measurements remained consistent, with deviations within ± 20% of nominal values throughout the study period, confirming the method’s reliability under refrigerated conditions in GuHCl. The dotted line represents +/− 20% deviation from the initial sample values (100%).

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The stability of mucus samples stored in 4 M GuHCl at 4 °C was assessed over 16 months. Figure 5B demonstrates that mucin concentrations remained consistent throughout the storage period, with fluctuations within the acceptable deviation limit of ± 20% from the baseline (day-zero) values (Fig. 5B, Supplementary Table 8). No significant degradation trends were observed over time, confirming the stability of mucin standards under refrigerated conditions in 4MGuHCl. These findings validate 4 M GuHCl as a suitable medium for the long-term storage of mucin standards at 4 °C.

Room temperature stability results indicated that the samples remained stable for 5 days. Short-term and long-term stability assessments in the fridge (2–8 °C) showed acceptable agreement with baseline (zero-day) results, lower than 10% of the nominal values (Supplementary table 8, supplementary Fig. 4).

The stability experiments demonstrated that the total mucin standard solution remained stable for up to 2 months within 10% retention and 9 months within 20%. However, the concentrations reduced substantially (more than 20%) after 10 months. The measurement time range is 16 months when stored at 2–8 °C (Supplementary Table 9, Supplementary Fig. 5).

To assess sample stability in the autosampler, mean values were calculated and compared with the zero-day results. All the results at 24, 48, and 144 h were lower than 10% of the nominal values (Supplementary Table 10).

Discussion

Research has established a clear link between increased mucin and mucus concentrations and symptoms, disease progression, and exacerbations in chronic respiratory conditions, including COPD, asthma, CB, CF, and NCFB1,3,10,11,13. Notably, the increased concentration of mucins is associated with the initiation and progression of COPD, as defined by the GOLD spirometric criteria, which currently depends on pulmonary lung function tests. Therefore, accurate mucin quantification in lung samples is critical for understanding disease initiation and progression and improving patient management.

There are two different but complementary approaches to mucin quantitation. First, total mucin concentration measurement: This represents the bulk quantity of large mucin polymers, which dominate the osmotic pressure of the mucus layer and govern the mucus transport on airway surfaces10,16. This measurement is achieved by SEC-MALLS-dRI or SEC-dRI, which is the focus of this study for analytical validation.

Second, individual mucin quantification: The two major secreted airway mucins, MUC5AC and MUC5B, that contribute to total airway mucin, but each has distinct biological functions17. Current state-of-the-art techniques, such as LC–MS/MS8,9, are replacing the older antibody-based methods due to their greater accuracy and sensitivity. Quantifying each mucin individually and analyzing their ratios provides additional insights into the lung in health and disease states.

We previously reported that total mucin levels at severe COPD were increased two to three-fold as compared to the non-smoker control group, with a progressive increase observed from stage 1 to stage 33. We also demonstrated that mucin concentrations were significantly higher in ever smokers who have no COPD, the so-called at-risk group, compared to never-smoked health controls. Furthermore, COPD patients experiencing frequent exacerbations (two or more annually) exhibited nearly double the mucin levels compared to those with fewer exacerbations. Elevated mucin levels were also significantly higher in patients with NCFB13 and CF compared to healthy subjects11,12.

Given the role of mucin as both a diagnostic/prognostic marker and a potential therapeutic target, its accurate quantification in clinical sputum samples (induced or spontaneous) is important. It’s crucial to highlight that currently, no objective biomarker is available for diagnosing CB, making the total mucin measurement presented here a novel approach for use in healthcare and research settings. Currently, the diagnosis of CB relies on the SGRQ or classic ATS questionnaires asking about their sputum and cough production and their quality of life18,19. We have demonstrated airway total mucin concentrations as a marker of CB and COPD3, offering a novel approach to diagnosing and monitoring this condition. We later showed that other chronic obstructive lung disease total mucins also could be informative about the severity of the disease11,13.

To help the effort of using this method routinely in the clinic for diagnosis and prognosis of mucoobstructive lung diseases and in clinical trials to test the effectiveness of the therapies, in this study, the SEC-(MALLS)-dRI method for quantifying total mucin was validated, achieving robust, accurate and precise analytical results.

The within-run and between-day precision studies demonstrated variation no greater than 2.6% and 2.32% CV, respectively. A comparison of total mucin levels measured using two different in-house instruments (with manual- and auto-injection) revealed a minimal difference of 2.11% (Fig. 1c), highlighting the precision of the SEC-(MALLS)-DRI method. The accuracy of this method was further supported by a recovery study, which found total mucin recovery to be 116%. A parallelism study was conducted to assess the effect of sample dilution, establishing the method’s relative accuracy. The method’s linearity was confirmed across a wide range of concentrations (30.49 to 3947.42 µg/mL), with a correlation coefficient (r^2) of 0.9947, indicating the robustness of the SEC-(MALLS)-dRI method.

The quantitative study of mucins in induced or spontaneous sputum samples inherently presents unique challenges, particularly in sample collection and processing. This is due to the potential for saliva contamination, and mucin’s inherently complex, viscous nature. To ensure reliable results, it is critical to provide patients with clear instructions for sputum collection and processing for subsequent mucin quantitation. Nationwide COPD study SPIROMCS20 and early COPD study SOURCE21 have implemented a standardized protocol for sputum induction and post-processing for mucin analysis. The simplicity of the sample preparation process, which notably does not require extraction, minimizes potential analytical errors.

In the context of highly inflammatory diseases such as CF, DNA contamination in sputum samples is a significant concern. However, in COPD sputum samples the DNA interference is less than 2% and does not affect the mucin measurements. The presence of high DNA concentrations in CF sputum samples due to high neutrophilic inflammation, however, can interfere with mucin measurement, underscoring the necessity of employing DNase treatment to ensure the accuracy of mucin quantification. This study specifically addressed the potential for DNA contamination, demonstrating that while there is no significant interference at lower DNA concentrations, interference becomes marked at higher levels. Therefore, the use of DNase treatment is highly recommended for samples obtained from highly inflammatory disease conditions such as CF to mitigate this interference and enhance the reliability of mucin measurements. While our findings provide insight into potential interference effects of DNA, saliva, and blood, further studies are needed to determine how these thresholds apply to real-world clinical samples.

The lower limit of quantitation (LLOQ) was determined to be 29.19 µg/mL, and the upper limit of quantitation (ULOQ) was established at 14,525  µg/mL, with the method demonstrating sensitivity at the LLOQ through consistent light-scattering detection. Stability studies demonstrated that clinical mucus samples stay stable for up to 16 months at 4 °C in 4MGuHCl up to 10 months at − 20 °C. The mucin standards remain stable for up to 8 months when refrigerated in 4MGuHCl. The method also exhibited reliable performance through freeze–thaw cycles and during long-term storage, further underscoring its suitability for use in clinical laboratories.

Clinical and research implications

Total mucin quantitation offers significant promise as a practical diagnostic and prognostic biomarker. Elevated mucin concentrations are strongly associated with disease severity, exacerbation frequency, and poor clinical outcomes in COPD, asthma, CF, and NCFB. In these conditions, the overproduction and hyperconcentration of airway mucins play a critical role in airway obstruction and exacerbations. While spirometry and symptom-based questionnaires remain the cornerstone of the diagnosis for CB, COPD, and asthma, they fail to provide direct, objective insights into mucus dynamics. The SEC-(MALLS)-dRI or SEC-dRI method bridges this gap by providing an objective, biological quantitative measure of airway mucus burden, which may help stratify patients with high-risk mucus phenotypes and adapt treatment strategies accordingly. By providing an objective biomarker, this method has the potential to complement traditional tools such as the SGRQ, Asthma Control Test (ACT), and spirometry, improving diagnostic accuracy and enabling earlier identification of at-risk populations.

Additionally, total mucin quantitation could serve as a critical endpoint in clinical trials evaluating therapies aimed at reducing mucus burden. For example, the use of mucolytics, CFTR modulators, or targeted anti-mucin therapies and biologics targeting T1 and T2 inflammations could be monitored more effectively through objective mucin measurements, enhancing the precision of efficacy assessments.

In conclusion, in this study, we present the comprehensive analytical validation of the SEC-(MALLS)-dRI method for quantifying total mucin concentrations in induced or spontaneous sputum samples. The technique demonstrates excellent precision, accuracy, and linearity, with a broad dynamic range that accommodates the variability across patient populations. Stability testing confirms its robustness for long-term sample storage, while the absence of carry-over ensures reliability in high-throughput workflows.

The clinical implications of this work are profound. By providing a standardized, objective tool for measuring total mucin concentrations, this method addresses a long-standing gap in diagnosing and monitoring hypersecretory hyperinflammatory lung diseases. It offers clinicians and researchers a reliable biomarker to stratify disease severity, assess exacerbation risk, and evaluate therapeutic responses. This is particularly relevant in asthma, COPD, and asthma COPD overlap (ACO), where mucus hypersecretion exacerbates symptoms and contributes to airway obstruction with different and common etiologies, and total mucin measurements could inform both diagnosis and management.

With its demonstrated robustness and clinical utility, this method has the potential to revolutionize the management of CB, COPD, asthma, and other muco-obstructive conditions. By enabling earlier intervention, patient stratification, and better therapeutic monitoring, the SEC-(MALLS)-dRI method could significantly improve patient outcomes and advance respiratory medicine.

Materials and methods

Additional details on material methods and validation strategies can be found in the Supplementary Appendix.

Induced sputum samples

Induced and spontaneous sputum samples used to validate this method were leftover, pooled, and uncoded samples from multiple previous studies3,11,13. The studies from which the sputum samples originated were conducted in accordance with relevant guidelines and regulations, including the Declaration of Helsinki. Sputum collection and processing protocols were approved by relevant institutional review boards (IRBs), and ethical clearance was obtained prior to sample collection. Informed consent was obtained from all participants at the time of the original studies, allowing the use of specimens for future research purposes. As the samples used in this study were de-identified, no additional ethical approval was required for their use in method validation.

Typically, these samples were obtained using a standard induction protocol20, kept on ice, and processed within 2 h. The samples were then transferred to sterile polypropylene centrifuge tubes, mixed with 8 M GuHCl (1:1, v/v), and stored at 2–8 °C until analysis.

Mucin QC standard

MUC5AC and MUC5B mucins, serving as the mucin standard, were isolated and purified from Calu3 or A549 monomucin cell cultures using a two-step process as previously described17. The isolated mucins were then dialyzed against deionized water and freeze-dried. The purified mucins were weighed, and a known amount was resuspended in a known volume of 4 M GuHCl buffer (0.1% EDTA, pH: 7.0) to be used as a standard at known concentrations.

Size Exclusion Chromatography coupled with MultiAngle Laser Light Scattering and differential Refractive Index detectors [SEC-(MALLS)- dRI] method was performed as described previously3,12. Briefly, samples were chromatographed on a Sepharose CL-2B size exclusion column (5 × 2.5 cm) to separate mucins from other proteins and eluted with 0.2 M NaCl containing 10 mM EDTA at a 500 μl/min flow rate. The column effluent was passed through in-line detectors, a laser photometer (MALLS, Dawn Heleos II; Wyatt Technology) coupled to a refractometer (dRI, Optilab; Wyatt Technology) to measure sample concentrations. Molecular size, determined by light scattering, was used to identify and define the mucin peak. While MALLS detection is not required for absolute mucin quantification, it is useful for confirming the presence of high-molecular-weight mucins, which is why it is included in parentheses (MALLS) in the method description. The resulting data were captured and analyzed using Astra software (Version 7.1., https://www.wyatt.com/products/software/astra.html, Wyatt Technology). Absolute mucin concentrations were calculated using differential refractometry, which measures the specific refractive index increment (dn/dc), reflecting the deviation of the refractive index by concentration. A dn/dc of 0.165 ml/g was used for mucins12.

Validation strategy

The SEC–(MALLS)-dRI for mucin measurement was validated following established guidelines15,22,23, covering precision, recovery and parallelism, specificity-selectivity and carry-over, linearity, analytical measurement range, quantitation limits, and stability. Unless otherwise stated, all experiments were performed in triplicate (n = 3) for each condition.

Precision was assessed under within-run (intra-assay), between-day, and within-laboratory conditions. For within-run precision, three mucin concentrations (high, medium, and low) were measured in nine replicates per batch. Between-day precision involved daily sample preparation and measurements of low-concentration standards over 10 days. Within-laboratory precision compared manual and automated sample injections using seven replicates each. Coefficients of variation (CV%) were calculated to evaluate variability.

Recovery and Parallelism: Recovery was evaluated by spiking induced sputum samples with known mucin concentrations and calculating recovery percentages: (Final concentration − Initial Concentration)/Added Concentration × 100%.

Parallelism was assessed across five serial dilutions of two clinical samples, comparing observed versus expected concentrations to confirm assay accuracy. The percentage recovery was determined by [Observed Concentration]/[Expected Concentration after dilution] × 100%.

Specificity-selectivity and carry-over

Specificity was tested by spiking sputum samples with potential contaminants, including saliva, blood, and DNA. Interference thresholds were determined by comparing mucin concentrations in spiked and unspiked samples. Also, DNA interference was mitigated by DNase treatment. Carry-over was evaluated by injecting blank samples after high-concentration samples, ensuring no residual mucin peaks were detected. All interference experiments were performed in triplicate (n = 3) for each condition.

Linearity, analytical measurement range, lower and upper limit of quantitation

Linearity was evaluated in two independent experiments conducted on different days and different concentration ranges. The first experiment assessed nine concentration levels (~ 30 to ~ 4000 µg/ml), while the second covered seven concentration levels (~225–14,500 µg/ml). The lower limit of quantitation (LLOQ) and upper limit of quantitation (ULOQ) were determined based on CV% and accuracy criteria.

Stability

Stability assessments were conducted for mucin standards and pooled induced sputum samples under various storage conditions. To ensure reproducibility, all stability experiments were performed in triplicate (n = 3) for each condition.

Standard stability

Mucin standards in 4 M GuHCl were stored at 2–8 °C for up to 13 months. Short-term stability was evaluated on days 1–4, while long-term stability was assessed weekly for the first month, bi-weekly for two months, and monthly thereafter. Stability was determined by comparing measured concentrations to the baseline (day-zero), with deviations ≤ 20% considered acceptable.

Sample stability

The mean values of triple measurements at each time point were compared to baseline, with acceptable deviations of ≤ 20%.

  • Frozen stability (− 20 °C) Samples were aliquoted and stored for up to 16 months. Freeze–thaw stability was assessed after two cycles (24-h freeze, room-temperature thaw).

  • Refrigerated stability (2–8 °C) Samples in 4 M GuHCl were monitored monthly for 16 months.

  • Room temperature stability Samples were tested over a 5-day benchtop period.

Autosampler stability

Samples stored in a 2–8 °C autosampler were re-injected at 24, 48, and 144 h to confirm stability. Deviations ≤ 10% were considered acceptable.

Statistical analysis

Where applicable, the statistical analyses were performed using the unpaired samples t-test.