A New Matrix Certified Reference Material for Measurement of Chlormequat Chloride and 2,4-Dichlorophenoxyacetic Acid Residues in Cucumber

Abstract

A novel cucumber pulp certified reference material (CRM) was prepared for the analysis of plant growth regulator residues, specifically chlormequat chloride (CCC) and 2,4-dichlorophenoxyacetic acid (2,4-D). The matrix CRM candidates were prepared by cucumber pulping, spiking, homogenizing and subpackaging. A reference method of liquid chromatography tandem isotope dilution mass spectrometry (ID-LC-MS/MS) was established for simultaneous measurement of mass fractions of CCC and 2,4-D in cucumber pulp. Interlaboratory value assignment of CCC and 2,4-D in the cucumber pulp CRM was performed using the ID-LC-MS/MS method. The certified values with expanded uncertainties (coverage factor k = 2) were assigned to be 4.1 mg/kg ± 0.4 mg/kg for CCC, 2.0 mg/kg ± 0.2 mg/kg for 2,4-D. Homogeneity assessment was performed on fifteen randomly selected units, and statistical analysis confirmed the CRM’s homogeneity for both CCC and 2,4-D, both between and within packages. The long-term stability of the CRM at −20 °C storage condition and short-term stability at 30 °C were monitored for 14 months and nine days, respectively, and no significant trend differences were observed. The uncertainty contributions from characterization, homogeneity and stability were taken into account in uncertainty evaluation. The CRM was officially certified and registered under the number GBW(E)100932 by the State Administration for Market Regulation of the P. R. China.

1. Introduction

Plant growth regulators (PGRs) are a class of natural or artificially synthesized compounds that modulate various physiological processes in plants at extremely low concentrations. Since their systematic application in agricultural production began in the 1930s, they have become an indispensable input in modern agriculture worldwide [1]. Significant research progress and considerable application results have been achieved in regulating crop growth, increasing yield, and improving quality [2]. However, many PGRs exhibit subacute or chronic toxicity, and their residues in agricultural products, such as fruits and vegetables, may pose potential risks to human health [3,4]. Therefore, controlling and regulating the use of PGRs in crop production and monitoring their residue levels are crucial for ensuring food safety. Chlormequat chloride (CCC) is a quaternary ammonium plant growth regulator that can shorten stems, thicken stalks, and deepen leaf color, thereby effectively controlling plant growth and promoting yield and quality [5,6]. However, improper use of CCC may lead to adverse effects on mammalian fertility [7,8]. The potential harm of CCC to humans and animals has garnered increasing attention from countries and institutions worldwide. Notably, the National Institute for Occupational Safety and Health (NIOSH) in the United States has classified CCC as a suspected endocrine disruptor [9]. 2,4-dichlorophenoxyacetic acid (2,4-D) is a synthetic auxin analog that can prevent fruit abscission. However, it is potentially carcinogenic and mutagenic in humans and livestock, and it is known to disrupt the endocrine, reproductive, and immune systems [10,11,12]. Due to these risks, many countries and regions have established maximum residue limits (MRLs) for CCC and 2,4-D in agricultural products to ensure food safety.

For instance, China has issued the national standard GB 2763-2021, “National Food Safety Standard—Maximum Residue Limits for Pesticides in Food,” which sets MRLs for CCC and 2,4-D in fruits and vegetables at 1.0 mg/kg and 0.5 mg/kg, respectively [13]. Similarly, the Codex Alimentarius Commission (CAC), the European Union (EU), and Japan have also established their own limits for CCC. For 2,4-D in cucumber, specific MRLs have been set by the EU at 0.05 mg/kg [14], the United States (US) at 0.1 mg/kg [15], and Japan at 0.08 mg/kg [16]. Several analytical methods are available for the detection of CCC and 2,4-D, including high-performance liquid chromatography (HPLC) [17], gas chromatography-tandem mass spectrometry (GC-MS) [18], and liquid chromatography-tandem mass spectrometry (LC-MS) [19,20,21]. Ensuring the accuracy of these methods is therefore critical for the reliability of test results. The use of matrix-certified reference materials (CRMs) is a highly effective approach for this purpose. With the rapid advancement of CRM development, various matrix CRMs have been produced, such as those for inorganic elements in coffee beans [22], zearalenone and its metabolites in corn flour [23], and toxic elements in cannabis leaves [24].

However, a significant gap remains in the availability of appropriate matrix CRMs to ensure accurate and reliable measurement of CCC and 2,4-D residues in food matrices. This deficiency underscores the urgent need for the development of such CRMs. Unlike existing pesticide CRMs for soil or fruit matrices [25], this study focuses on a high-water-content cucumber matrix. Following ISO Guide 35:2017 [26] for homogeneity, stability, and characterization, we developed a new certified reference material specifically for laboratories monitoring CCC and 2,4-D residues in cucumber. An isotope dilution-liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) method was established as the reference method and applied for cooperative value assignment among participating laboratories. The ID-LC-MS/MS method was employed for both homogeneity testing and stability monitoring. In addition, a systematic analysis and evaluation of the uncertainties arising from the CRM development process was performed.

2. Experimental Section

2.1. Material and Methods

The pure certified reference material (CRM) for 2,4-D (GBW09291, purity 99.6%) was provided by the Institute of Quality Standard and Testing Technology for Agro-Products (IQSTAP), Chinese Academy of Agricultural Sciences (CAAS) (Beijing, China). A CCC solution CRM (GBW(E)081910, 100 mg/L) was obtained from Beijing Coastal Hongmeng Standard Material Technology Co., Ltd. (Beijing, China). Isotopically labeled internal standards, CCC-D4 (>98% purity) and 2,4-D-D3 (>98% purity), were purchased from CDN Isotopes, Pointe-Claire, QC, Canada. HPLC-grade methanol and acetonitrile were sourced from Merck (Darmstadt, Germany), and formic acid (99% purity) was supplied by Beijing J&K Chemical Technology Co., Ltd. (Beijing, China). Deionized water was obtained from Wahaha Co., Ltd., Beijing, (China).

An ultra-high performance liquid chromatography system (Acquity, Waters, Milford, CT, USA) coupled with an AB Sciex API 4500 mass spectrometer (AB Scuex, Framingham, MA, USA) was used for the analysis. Chromatographic separation of CCC and 2,4-D from the sample matrix was achieved using a Waters X-bridge C18 column (150 mm × 2.1 mm, 3.5 μm). Sample preparation was performed using a Vortex Mixer VORTEX-5 (Scientific Industries, Bohemia, NY, USA). For the preparation of standard solutions and the weighing of samples, analytical balances AL106 (d = 0.1 mg), XS105DU (d = 0.01 mg), and XP6 (d = 0.001 mg) (Mettler-Toledo, Greifensee, Switzerland) were employed.

2.2. Standard Solution Preparation

A stock standard solution of 2,4-D (100 mg/kg) was prepared using the gravimetric method. Isotope-labeled standard solutions of CCC-D4 and 2,4-D-D3 (500 mg/kg each) were also prepared. All standard solutions were stored in sealed brown ampoule foil bags at −20 °C and were stable for up to 6 months. The mass fractions of the working solutions were 100 mg/kg for CCC and 40 mg/kg for 2,4-D. A 1:1 amount ratio of CCC to CCC-D4 and of 2,4-D to 2,4-D-D3 was maintained for all analyses.

2.3. Preparation of Certified Reference Material Candidates

To ensure traceability, a single batch of fresh cucumbers was purchased from a local supermarket in Beijing, China. Cucumbers of uniform size (approximately 15 cm in length), similar ripeness, and with no surface blemishes were selected. After washing with purified water, the cucumbers were diced into small pieces. Prior to use, a subsample was analyzed to confirm the absence of detectable residues of CCC and 2,4-D, thereby ensuring that the matrix was free of the target analytes. Approximately 15 kg of the diced cucumber was homogenized in a high-speed blender (Burley R6201B, Burley Tools, Hangzhou, Zhejiang, China) for 2 min and then transferred into a clean 20 L white plastic bucket. Pre-pared solutions of CCC and 2,4-D were subsequently added to the cucumber pulp. The bucket was sealed and then shaken and rolled for 40 min to ensure thorough homogenization of the analytes with the cucumber matrix. Next, 5 kg of this homogenized pulp was transferred to a clean stainless steel bucket and mixed for an additional 20 min. Following homogenization, the bulk material was dispensed into food-grade aluminum foil bags under continuous stirring, with each bag containing 20 g, yielding a total of 200 units. Finally, the CRM candidates were stored at −20 °C. No antimicrobial agents were added to avoid potential matrix interference; therefore, immediate use after thawing is recommended.

2.4. Sample Pretreatment

A 2.00 g portion of cucumber pulp (weighed to an accuracy of 0.01 g) was transferred into a 50 mL centrifuge tube. An appropriate volume of isotope internal standard solution was then added, and the tube was weighed again to record the exact mass added. Prior to subsampling, the CRM candidate was thawed at 4 °C, vortexed thoroughly at 2500 r/min for 5 min to ensure uniformity after possible syneresis, and then allowed to equilibrate at room temperature for 30 min [27]. Subsequently, 25 mL of acetonitrile containing 1% formic acid was added. After extraction and centrifugation, the supernatant was filtered through a 0.22 μm filter and analyzed by LC-MS/MS.

2.5. ID-LC-MS/MS Measurement

The chromatographic separation was performed using a Waters XBridge C18 column(Waters, Milford, CT, USA) (150 mm × 2.1 mm, 3.5 µm). The mobile phase consisted of (A) water containing 0.2% acetic acid and 5 mM ammonium acetate, and (B) acetonitrile containing 0.2% acetic acid. The elution program is detailed in Table S1. Mass spectrometry detection was carried out using an electrospray ionization (ESI) source in positive/negative switching mode with multiple reaction monitoring (MRM). Key instrument parameters were as follows: source temperature, 150 °C; capillary voltage, ±3.5 kV; desolvation gas temperature, 500 °C; desolvation gas (nitrogen) flow rate, 900 L/h; and collision gas (argon) flow rate, 0.16 mL/min. Additional MS parameters are provided in Table S2. Data acquisition was performed using Analyst 1.6.3 (SCIEX), quantitative analysis was conducted with MultiQuant 3.0, and statistical analysis was carried out using Microsoft Excel 2019.

2.6. Characterization Methodology

In accordance with the technical specification JJF 1343–2022, “Characterization, Homogeneity and Stability Assessment of Reference Materials,” the characterization of CCC and 2,4-D mass fractions in cucumber pulp was conducted by eight authoritative collaborating laboratories. The leading laboratory provided each participating laboratory with the necessary standard solutions, isotope-labeled internal standard solutions, and randomly selected CRM units (three units per laboratory) for the measurement. For the characterization, three subsamples from each CRM unit were analyzed using the ID-LC-MS/MS reference method to determine the mass fractions of CCC and 2,4-D.

2.7. Assessment of Homogeneity and Stability Methodology

For homogeneity assessment, 15 units of the CRM candidate were randomly selected, numbered from 1 to 15, and analyzed in triplicate using the ID-LC-MS/MS method. The data were evaluated by one-way analysis of variance (ANOVA) at a 95% confidence level. Based on these results, the minimum sample intake validated for this CRM is 2.0 g. Users should not use a sample size smaller than 2.0 g, as homogeneity and the certified uncertainty have only been verified for this intake.

For stability testing, the long-term stability of the CRMs was assessed at −20 °C using the ID-LC-MS/MS method. At predetermined time points (0, 1, 2, 3, 4, 6, 11, and 14 months), three units were randomly sampled, and three subsamples from each unit were analyzed. The stability monitoring data were evaluated using regression analysis. For short-term stability assessment, samples were stored at 4 °C and 30 °C, and stability monitoring was performed on days 0, 1, 3, 5, 7, and 9. The determination and analysis methods were the same as those used for long-term stability monitoring.

3. Results and Discussion

3.1. Preparation of CRM Candidates

Certified reference materials (CRMs) with a matrix composition matching that of real samples are widely used for method validation and quality control, as their properties closely resemble those of actual test materials. Furthermore, candidate matrix CRMs must be representative, with property values at concentrations that meet the requirements of practical testing applications. In China, the national standard GB 2763-2021 [13], “National Food Safety Standard—Maximum Residue Limits of Pesticides in Food,” establishes regulatory limits for certain plant growth regulators (PGRs). Specifically, the MRLs for CCC and 2,4-D in common fruits and vegetables are set at 1 mg/kg and 0.5 mg/kg, respectively. To ensure the applicability of the developed matrix CRMs for method validation and quality control in real sample testing, the target concentrations were set at 3–5 times the detection limit, based on prior experience in matrix CRM development. Accordingly, the residual concentrations of CCC and 2,4-D in the cucumber pulp were targeted to be within 3–5 mg/kg and 1.5–2.5 mg/kg, respectively, ensuring that the resulting CRMs are fit for their intended purpose.

Studies on PGRs have shown that while low concentrations exhibit beneficial effects on plants and fruits, such residues degrade rapidly during plant growth and cannot meet the required concentration ranges for matrix CRMs. Conversely, high-concentration applications may inhibit plant development or even cause plant mortality, making field-treated samples impractical for raw material acquisition. To address these challenges, this study adopted a laboratory-based approach to prepare qualified candidate materials. The preparation process involved pulping of the matrix, precise addition of the target PGRs, homogenization, sealed packaging, and low-temperature preservation. Prior to the preparation of the candidate reference materials, we systematically evaluated homogenization efficiency at different mixing durations (5, 10, 20, and 40 min). For both CCC and 2,4-D, the standard deviations of the measurement results decreased significantly with extended mixing time (Supplementary Table S3). Based on these observations, a 40 min mixing duration was established as the optimal parameter for homogenization.

The freeze–thaw stability of the CRM candidate was evaluated over three cycles, each consisting of thawing at 24 °C for 6 h followed by refreezing. Results from three units (Table S4) showed that after three cycles, the concentrations of CCC and 2,4-D remained stable, with small standard deviations (CCC: 0.04–0.08 mg/kg; 2,4-D: 0.01 mg/kg). This indicates no significant effect under standard operating conditions (≤3 cycles with vortexing before use).

3.2. Sample Preparation and ID-LC-MS/MS Analysis

3.2.1. Optimization of Sample Pre-Treatment

Numerous studies have reported that methanol, acetonitrile, acidified methanol, and acidified acetonitrile are effective for the extraction of plant growth regulators [28]. In the present study, the extraction efficiencies of these solvents for CCC and 2,4-D were evaluated, as shown in Figure S1a,b. The comparative analysis demonstrated that, overall, acidified organic solvents provided better extraction performance than their non-acidified counterparts, with acidified acetonitrile showing slightly superior extraction efficiency compared to acidified methanol. Therefore, 1% acidified acetonitrile was ultimately selected as the extraction solvent.

Simultaneously, the extraction conditions were optimized by investigating the effect of different extraction volumes (15, 25, and 50 mL). As illustrated in Figure S1c for CCC and Figure S1d for 2,4-D, a volume of 25 mL was found to be sufficient for adequate extraction. Additionally, the addition of sodium chloride is known to reduce the solubility of plant growth regulators in water, thereby enhancing extraction efficiency. Accordingly, the amount of sodium chloride added was optimized, and it was found that 2 g of sodium chloride improved extraction efficiency.

Finally, the effects of commonly used sorbents—PSA, C18, and GCB—on sample cleanup were evaluated. As shown in Figure S1e,f, while these purification agents did not significantly affect the measurement accuracy of the target analytes, their addition did result in some adsorption loss. Given the high concentration levels of the target analytes in the cucumber pulp, the sample pretreatment was simplified to extraction, centrifugation, filtration, and direct injection for instrumental analysis.

3.2.2. ID-LC-MS/MS Condition Optimization

The LC-MS/MS conditions were optimized for the analysis of CCC and 2,4-D. The results showed that CCC readily acquired protons in positive ion mode, forming the molecular ion [M+H]⁺; therefore, positive ion mode was selected for its detection. Conversely, 2,4-D tended to lose protons in negative ion mode, forming the more stable deprotonated ion [M-H]⁻, which justified the choice of negative ion mode for its detection. Chromatograms of CCC and 2,4-D, along with their corresponding isotopically labeled internal standards, in both calibration solution and cucumber sample matrix, are presented in Figure 1. First, the linearity of the method was assessed by analyzing a series of standard solutions. The correlation coefficients (R²) were 0.9990 for CCC and 0.9991 for 2,4-D, indicating excellent linearity within the respective concentration ranges and validating the suitability of the single-point calibration method. Next, the accuracy and precision of the method were evaluated using ID-LC-MS/MS with single-point calibration. The results (Supplementary Tables S5 and S6) demonstrated that both inter- and intra-group relative standard deviations (RSDs) were predominantly around 5%, indicating satisfactory measurement repeatability and precision.

In the recovery study, cucumber pulp matrix samples were spiked with CCC and 2,4-D at low, medium, and high concentration levels. The results (Supplementary Table S7) showed that the recoveries for CCC ranged from 100.4% to 101.1%, and for 2,4-D from 97.0% to 105.1%. These recovery and accuracy results met the requirements for characterization. Furthermore, the matrix effects (ME) for CCC, 2,4-D, and their isotopically labeled internal standards were evaluated. As shown in Supplementary Table S8, the ME values for all four compounds were approximately 1, indicating a negligible matrix effect. Moreover, the similar ME values for each analyte and its corresponding internal standard demonstrate that the use of isotope internal standards effectively compensates for any matrix effects, ensuring measurement accuracy. The limit of detection (LOD) and limit of quantification (LOQ) were defined as signal-to-noise ratios of 3 and 10, respectively. Under these criteria, the method achieved LOD/LOQ values of 1.26/4.19 μg/kg for CCC and 2.67/8.91 μg/kg for 2,4-D.

3.3. Characterization

Table 1. Measurement results of the eight participating laboratories (mg/kg).

Laboratory	CCC		2,4-D
	Mean (=9)	Standard Deviation (n = 9)	Mean (=9)	Standard Deviation (n = 9)
1	3.88	0.10	1.97	0.08
2	4.22	0.07	2.05	0.04
3	4.01	0.13	1.88	0.05
4	4.40	0.12	2.00	0.06
5	4.43	0.13	2.18	0.05
6	4.10	0.11	1.90	0.06
7	3.95	0.02	2.00	0.04
8	4.12	0.09	1.82	0.06
Overall mean	4.1		2.0
Overall standard deviation	0.2		0.1

presents the measurement data submitted by each participating laboratory. The Dixon test was employed to statistically identify outliers. As shown in Supplementary Table S9, the calculated r₁ and rₙ values for all laboratories were less than the critical value f_{(0.05, 9)}, indicating that no outliers were detected and all results were deemed acceptable. Subsequently, the Cochran test was applied to evaluate the consistency of measurement variances among laboratories at a significance level α = 0.05. The calculated C values for CCC and 2,4-D were 0.2001 and 0.2687, respectively, both below the critical value C_{(0.05, 8, 9)} = 0.3043. Therefore, the variances across the eight laboratories were considered to be homogeneous. Finally, the Dixon test was reapplied to check for outliers in the laboratory means, and none were found. The certified values for CCC and 2,4-D in the cucumber pulp were assigned as 4.1 mg/kg and 2.0 mg/kg, respectively, calculated as the mean of the means from all laboratories. The certified concentrations of CCC (4.1 mg/kg) and 2,4-D (2.0 mg/kg) in this CRM are above their respective maximum residue limits (MRLs) for cucumbers (1.0 mg/kg and 0.5 mg/kg). This concentration range (approximately 3–5 times the limit of detection) was intentionally selected to ensure satisfactory homogeneity and stability during CRM production, making the material suitable for method validation at moderate concentration levels. To further assess the robustness of the assigned values, robust statistics were calculated following the approach recommended in ISO 13528 [26], using the median and normalized median absolute deviation (MADn). Based on all individual measurement results (n = 72 for each analyte), the median value for CCC was 4.125 mg/kg with a MADn of 0.200 mg/kg, and the median for 2,4-D was 1.96 mg/kg with a MADn of 0.104 mg/kg. These robust estimates are in excellent agreement with the arithmetic means (4.14 mg/kg for CCC and 1.98 mg/kg for 2,4-D) and their corresponding standard deviations (0.21 mg/kg and 0.12 mg/kg, respectively). The consistency between the classical and robust statistics confirms that the assigned certified values (4.1 mg/kg for CCC and 2.0 mg/kg for 2,4-D) are reliable and not unduly influenced by potential outliers.

3.4. Assessment of Homogeneity and Stability

The mass fractions of CCC and 2,4-D within and between foil bags are shown in Figure 2a and Figure 2b, respectively. The homogeneity test results were statistically analyzed using one-way ANOVA. As shown in Supplementary Tables S10 and S11, the calculated F values were less than the critical F value, indicating that the homogeneity of the CRM candidates was acceptable. Additionally, the sample intake used for the homogeneity test was 2 g. Therefore, 2 g was established as the minimum sample intake for this matrix CRM. The uncertainty introduced by inhomogeneity, denoted as u_bb, encompasses variations arising from the sample processing procedure, the sub-sampling process, and the between-unit heterogeneity. This inhomogeneity component is quantified by the relative standard deviation of the homogeneity test results obtained between and within the foil bags.

To date, no studies have documented the stability of CCC and 2,4-D in cucumber pulp matrices. In this work, a comprehensive evaluation of their stability was conducted under conditions simulating long-term storage, transportation (short-term), and routine usage. The CRM was stored at −20 °C for 14 months, and the concentrations of CCC and 2,4-D were measured periodically by ID-LC-MS/MS (Figure 3a,b; Table S12). Trend analysis indicated that the slope of the regression line was not significantly different from zero, demonstrating acceptable long-term stability. To simulate transportation conditions, CRM samples were exposed to 4 °C and 30 °C for 1, 3, 5, 7, and 9 days. As shown in Figure 3c, the mass fractions of CCC remained within uncertainty limits throughout the study, and t-tests confirmed stability (calculated t-values < critical value). However, Figure 3d demonstrates that 2,4-D in cucumber pulp became unstable after 9 days of storage at 30 °C. Therefore, the transportation temperature for this CRM should not exceed 4 °C. The stability-related uncertainties consisted of components from long-term stability (u_lts) and short-term stability (u_sts). The observed instability of 2,4-D at 30 °C in the cucumber matrix, combined with the high water activity of this matrix, suggests that microbial degradation may be the primary mechanism. Papade et al. [29] demonstrated that Cupriavidus sp. DSPFs efficiently degrades 2,4-D at 30 °C (3000 mg·L⁻¹ within 24 h), indicating that this temperature supports the active metabolism of 2,4-D-degrading microorganisms. Tuan et al. [30] reported that high-moisture environments serve as hotspots for 2,4-D-degrading bacteria; the cucumber matrix similarly provides a favorable microenvironment for microbial activity. Notably, Dörfler et al. [31] clarified that 2,4-D acid is resistant to hydrolysis, suggesting that hydrolytic degradation contributes minimally in this system. Therefore, the instability observed at 30 °C is predominantly attributable to microbial degradation, facilitated by the high water activity of the cucumber matrix.

3.5. Uncertainty Assessment

The source of uncertainty originating from the development of certified reference material of CCC and 2,4-D in cucumber pulp mainly consists of three components: the uncertainty u_char that from the characterization of the certified reference material; the uncertainty u_bb from homogeneity; and the uncertainties u_lts and u_sts from the long-term stability and short-term stability, respectively. This can be calculated by the following formula:

$$u_{\mathrm{C}\mathrm{R}\mathrm{M},\mathrm{r}\mathrm{e}\mathrm{l}}=\sqrt{{u_{\mathrm{c}\mathrm{h}\mathrm{a}\mathrm{r},\mathrm{r}\mathrm{e}\mathrm{l}}}^2+{u_{\mathrm{b}\mathrm{b},\mathrm{r}\mathrm{e}\mathrm{l}}}^2+{u_{\mathrm{l}\mathrm{t}\mathrm{s},\mathrm{r}\mathrm{e}\mathrm{l}}}^2+{u_{\mathrm{s}\mathrm{t}\mathrm{s},\mathrm{r}\mathrm{e}\mathrm{l}}}^2}$$

$$U_{\mathrm{C}\mathrm{R}\mathrm{M}}=u_{\mathrm{C}\mathrm{R}\mathrm{M},\mathrm{r}\mathrm{e}\mathrm{l}}\times k\left(k=2\right)$$

where u_char represents the uncertainty arising from the characterization, and k denotes the coverage factor at the 95% confidence level. The uncertainty u_char is composed of two components: type A uncertainty arising from co-laboratory value assignment, and type B uncertainty associated with standard solution and sample preparation. The latter encompasses: (1) preparation of CCC and 2,4-D standard solutions; (2) mass of CCC and 2,4-D in calibration solutions; (3) mass of isotopically labeled CCC-D4 and 2,4-D-D3 in calibration solutions; (4) mass of isotopically labeled CCC-D4 and 2,4-D-D3 added to samples; and (5) sample weighing.

Table 2. Evaluation of uncertainty of CCC and 2,4-D in cucumber pulp CRM.

Uncertainty Source	Evaluation	CCC	2,4-D
Laboratories measurements, $u_{\mathrm{A},\mathrm{r}\mathrm{e}\mathrm{l}}$, (%)	$u_{\mathrm{A}}=\sqrt{{\displaystyle \frac{{\sum _{i=1}^n\left({\overline{x}}_i-\overline{\overline{x}}\right)}^2}{n\times \left(n-1\right)}}}$,$u_{\mathrm{A},\mathrm{r}\mathrm{e}\mathrm{l}}={\displaystyle \frac{u_{\mathrm{A}}}{\overline{x}}}$	1.72	1.77
$u_{\mathrm{B},\mathrm{r}\mathrm{e}\mathrm{l}}$, (%)	$u_{\mathrm{B},\mathrm{r}\mathrm{e}\mathrm{l}}=\sqrt{{u_{\mathrm{s}\mathrm{t}\mathrm{d},\mathrm{r}\mathrm{e}\mathrm{l}}}^2+{u_{\mathrm{c}\mathrm{a}\mathrm{l},\mathrm{r}\mathrm{e}\mathrm{l}}}^2+{u_{\mathrm{m}\mathrm{E}\mathrm{v}\mathrm{a}-\mathrm{d}\mathrm{4,2},4-\mathrm{D}-\mathrm{d}5,\mathrm{r}\mathrm{e}\mathrm{l}}}^2+{u_{\mathrm{s}\mathrm{a}\mathrm{m},\mathrm{r}\mathrm{e}\mathrm{l}}}^2}$	1.02	0.52
Characterization, $u_{\mathrm{c}\mathrm{h}\mathrm{a}\mathrm{r},\mathrm{r}\mathrm{e}\mathrm{l}}$ (%)	$u_{\mathrm{c}\mathrm{h}\mathrm{a}\mathrm{r},\mathrm{r}\mathrm{e}\mathrm{l}}=\sqrt{{u_{\mathrm{A},\mathrm{r}\mathrm{e}\mathrm{l}}}^2+{u_{\mathrm{B},\mathrm{r}\mathrm{e}\mathrm{l}}}^2}$	2.05	1.84
Homogeneity test, $u_{\mathrm{b}\mathrm{b},\mathrm{r}\mathrm{e}\mathrm{l}}$ (%)	$u_{\mathrm{b}\mathrm{b}}=S_{\mathrm{H}}=\sqrt{{\displaystyle \frac{s_1^2-s_2^2}{\mathrm{n}}}},u_{\mathrm{b}\mathrm{b},\mathrm{r}\mathrm{e}\mathrm{l}}={\displaystyle \frac{S_{\mathrm{H}}}{\overline{X}}}$	0.73	2.62
Long-term stability study, $u_{\mathrm{l}\mathrm{t}\mathrm{s},\mathrm{r}\mathrm{e}\mathrm{l}}$ (%)	$u_{\mathrm{l}\mathrm{t}\mathrm{s}}=s\left({\beta }_1\right)·t,u_{\mathrm{l}\mathrm{t}\mathrm{s},\mathrm{r}\mathrm{e}\mathrm{l}}={\displaystyle \frac{u_{\mathrm{l}\mathrm{t}\mathrm{s}}}{\overline{X}}}$	4.03	2.53
Short-term stability study, $u_{\mathrm{s}\mathrm{t}\mathrm{s},\mathrm{r}\mathrm{e}\mathrm{l}}$ (%)	$u_{\mathrm{s}\mathrm{t}\mathrm{s}}=s\left({\beta }_1\right)·t,u_{\mathrm{s}\mathrm{t}\mathrm{s},\mathrm{r}\mathrm{e}\mathrm{l}}={\displaystyle \frac{u_{\mathrm{s}\mathrm{t}\mathrm{s}}}{\overline{X}}}$	1.16	2.02
Relative combined uncertainty, $u_{\mathrm{C}\mathrm{R}\mathrm{M},\mathrm{r}\mathrm{e}\mathrm{l}}$ (%)	$u_{\mathrm{C}\mathrm{R}\mathrm{M},\mathrm{r}\mathrm{e}\mathrm{l}}=\sqrt{{u_{\mathrm{c}\mathrm{h}\mathrm{a}\mathrm{r},\mathrm{r}\mathrm{e}\mathrm{l}}}^2+{u_{\mathrm{b}\mathrm{b},\mathrm{r}\mathrm{e}\mathrm{l}}}^2+{u_{\mathrm{l}\mathrm{t}\mathrm{s},\mathrm{r}\mathrm{e}\mathrm{l}}}^2+{u_{\mathrm{s}\mathrm{t}\mathrm{s},\mathrm{r}\mathrm{e}\mathrm{l}}}^2}$	4.73	4.56
Relative expanded uncertainty, $U_{\mathrm{c},\mathrm{r}\mathrm{e}\mathrm{l}}$ (%)	$U_{\mathrm{c},\mathrm{r}\mathrm{e}\mathrm{l}}=u_{\mathrm{C}\mathrm{R}\mathrm{M},\mathrm{r}\mathrm{e}\mathrm{l}}\times k$	9.5	9.1
Coverage factor, k		2	2
Certified value, (mg/kg)		4.1	2.0
$U_{\mathrm{C}\mathrm{R}\mathrm{M}}$ (mg/kg)	$U_{\mathrm{C}\mathrm{R}\mathrm{M}}=U_{\mathrm{c},\mathrm{r}\mathrm{e}\mathrm{l}}\times C$	0.4	0.2

provides a summary of the uncertainty sources, calculation formulas and the corresponding evaluation results. The largest contribution to the combined uncertainty for CCC comes from long-term stability ($u_{\mathrm{l}\mathrm{t}\mathrm{s}}=s\left({\beta }_1\right)·t,u_{\mathrm{l}\mathrm{t}\mathrm{s},\mathrm{r}\mathrm{e}\mathrm{l}}={\displaystyle \frac{u_{\mathrm{l}\mathrm{t}\mathrm{s}}}{\overline{X}}}$ = 4.03%), reflecting the inherent variability in a high moisture matrix CRM over 14 months. This component remains within acceptable limits, as no significant degradation trend was observed (|β₁| < $t_{\left(0.95,4\right)}·s\left({\beta }_1\right)$), and the expanded uncertainty ($U_{\mathrm{C}\mathrm{R}\mathrm{M}}=U_{\mathrm{c},\mathrm{r}\mathrm{e}\mathrm{l}}\times C$ = 0.4 mg/kg) already covers this source, supporting the 14-month shelf life.

3.6. Greenness Assessment of the CRM Characterization Method

The greenness of the analytical method used for CRM characterization was evaluated using the AGREE metric [32]. This tool assesses analytical methods based on the 12 principles of green analytical chemistry, yielding an overall score between 0 and 1, where higher scores indicate more environmentally friendly procedures. The method achieved an overall score of 0.38 (Figure S2). The main limitations were the use of approximately 25 mL of acetonitrile per sample and the relatively large sample size of 2 g. While these conditions were optimized to ensure maximum recovery during CRM characterization, future users may consider exploring reduced solvent volumes or greener alternatives for routine analysis, provided appropriate validation is performed.

3.7. Limitations of Existing CRMs and the Matrix-Matched Advantages of This CRM

To highlight the specific advances of this work, the key features of existing reference materials for CCC and 2,4-D are summarized and compared with our CRM in Table S13. Currently available CRMs are limited to pure substance or solution forms, which are suitable for instrument calibration but cannot be used to validate sample preparation procedures or assess matrix effects. In contrast, our CRM is the first commutability-tested cucumber pulp matrix CRM that simultaneously certifies both CCC and 2,4-D, and it has been developed in full compliance with JJF 1343 (equivalent to ISO Guide 35). This matrix CRM provides a valuable tool for method validation, quality control, and proficiency testing in food safety laboratories.

4. Conclusions

This paper presents the development of a new matrix certified reference material (CRM) for the determination of CCC and 2,4-D residues in cucumber pulp. The CRM candidates, fortified with CCC and 2,4-D, exhibit physical properties and certified concentration values representative of real contaminated samples. These cucumber pulp matrix CRMs were prepared by spiking blank cucumber homogenate with the target analytes. The ID-LC-MS/MS method was employed for characterization by the participating laboratories. Homogeneity and stability assessments confirmed that the CRM remains homogeneous and stable for at least 14 months when stored at −20 °C, and for up to 9 days under 30 °C conditions. A systematic evaluation of uncertainties arising from the CRM development process was also performed. Certified as GBW(E) 100932 by the State Administration for Market Regulation of the P. R. China, this reference material provides an effective tool for method validation and quality control in the determination of CCC and 2,4-D residues in cucumber pulp. However, recognizing the need for CRMs at concentrations near regulatory limits, future work will focus on developing materials with mass fractions at or below the MRLs to support routine compliance testing.