Screening for contamination levels of select organic environmental chemicals in medical supplies used for human specimen collection

Abstract

Trace-level analysis of environmental chemicals in human specimens can be compromised by contamination introduced during sample collection and storage. Sampling devices and tools can be a source of sample contamination by plasticizers, additives and antimicrobials, which warrants the need for pre-screening of these products prior to use. In this study, we determined leaching of 121 environmental chemicals in 10% and 100% methanol from 24 types of human specimen collection and storage devices. Cryovials, serum tubes, cups, syringes, transfer pipettes, and gloves -commonly used for the collection of blood, urine, breast milk and stools - were screened for the presence of plasticizers, environmental phenols, and pesticides. Measurable concentrations of mono-ethyl phthalate (mEP) and triethyl phosphate (TEP) were leached from vials, plastic bags, gloves, and diapers, and parabens were leached in collection bottles, at concentrations exceeding 100 ng/ device. The amount leached from the devices varied depending on the lot numbers of the same product type. Storage time and temperature were found to influence the leaching rate of chemicals, with increased levels observed following prolonged storage and at high temperatures. The study underscores the importance of prescreening for contamination in devices used for collection and storage of human specimens for biomonitoring studies.

Graphical Abstract

1. Introduction

Accurate detection and quantification of trace levels of environmental chemicals present in human biospecimens are critical for assessing exposures and potential health risks (Dennis et al., 2017). For biomonitoring studies, biological specimens such as urine and blood must be collected and managed in a manner that preserves their quality and minimizes external contamination. However, among several factors, data quality can be compromised by inadvertent introduction of contaminants arising from devices used during biospecimen collection and storage (Ye et al., 2013). This issue is particularly critical when measuring picogram to nanogram levels of organic chemicals present in human specimens (Manz et al., 2022). Screening or lot testing of collection materials is important in biomonitoring studies because many target analytes (e.g., antimicrobial preservatives, additives, plasticizers) are ubiquitous in the environment. Lot screening is performed by measuring the concentration of the target analyte(s) in a screening solvent (i.e., water or a simulant) before and after contact with the device or container being tested (see Improving the Collection and Management of Human Samples Used for Measuring Environmental Chemicals and Nutrition Indicators (cdc.gov).

Many devices used for specimen collection and storage are made of plastic, rubber, synthetic fibers or other polymers that contain several target chemicals of interest in human biomonitoring studies. Nevertheless, studies that determine leaching of environmental chemicals from devices used for human specimen collection and storage are meager. Few studies have demonstrated the occurrence of chemicals such as phthalate esters in various plastic products, including solid-phase extraction cartridges, pipette tips, and vial-septa used during chemical analysis (Guo and Kannan, 2012, Yamashita et al., 2004). Linear alkylbenzene sulfonates were found in nitrile gloves used in laboratories (Li et al., 2020). Contamination of blood by bisphenol A (BPA) from butterfly vacutainer collection devices has been reported (Bousoumah et al., 2021, Cerkvenik-Flajs 2022). Similarly, contamination by trace elements in serum collection tubes has been documented (Choe et al., 2022).

Although majority of analytes measured in human biomonitoring studies are biological metabolites of environmental chemicals used in products (and therefore contamination introduced by collection materials of metabolites is less likely), for some chemicals, parent compounds are measured as biomarkers of exposure. For instance, organophosphate esters (used as plasticizers and flame retardants), parabens (antimicrobials) and bisphenols (plasticizers) are commonly measured as exposure biomarkers in urine. Occurrence of these classes of chemicals in consumer products, including medical devices, is known (Guo et al., 2014, Liao and Kannan 2014, Asimakopoulos et al., 2016, Xue et al., 2017, Gao and Kannan 2020, Zhu and Kannan 2020, Bernard et al., 2023, Wang and Kannan 2023).

Plasticizers such as phthalic acid esters (PAEs) and organophosphate esters (OPEs) are widely used in the manufacture of plastics to enhance their flexibility and durability (Huo et al., 2023, Wang and Kannan 2023). They are known for their potential endocrine-disrupting effects (Andersen et al., 2018). Environmental phenols such as bisphenols (e.g., BPA) and parabens are commonly used in consumer products as plastic additives and antimicrobial preservatives, respectively (Liao and Kannan 2018, Sol et al., 2023). These compounds have been linked to hormone disruption and potential carcinogenic effects (Helm et al., 2018, Kannan and Vimalkumar 2021). Pesticides are used extensively in agriculture and indoor pest control (Wang et al., 2019, Zhao et al., 2023).

The National Institutes of Health (NIH) of the United States launched the Environmental influences on Child Health Outcomes (ECHO) program in 2016 to understand the effects of a broad range of environmental influences on child health and development. The program aimed to achieve standardized biospecimen collection methods from over 50 cohorts comprising >50,000 children throughout the United States. Biospecimen collection, processing, and storage protocols for blood, urine, stool, and placenta samples from pregnant mothers and their children were developed by the program and the biorepository was managed by Fisher Bioservices (Rockville, MD, USA) (Blaisdell et al., 2022). The standardized sample collection protocols also comprised prescreening of supplies used in specimen collection and storage, for selected environmental chemicals. In this context, we developed a method to measure 121 environmental chemicals in urine for the ECHO program (Zhu et al., 2021) and that method was also applied for prescreening of collection devices and supplies. This study is focused on leaching of 121 environmental chemicals from cryovials, tubes, cups, syringes/needles, cotton pads, and diapers, used for collection and storage of urine, blood/serum, breast milk and stool (Buckley et al., 2022, Xie et al., 2022, Zhao et al., 2023b). Additionally, gloves used for personal protection and plastic bags used for specimen storage were tested for contamination. These supplies, typically made of polypropylene/polyethylene plastic or synthetic/cotton fabric may contain a range of chemicals, including plasticizers and antimicrobials (Chen et al., 2022, Vimalkumar et al., 2022).

In this study, we developed protocols to study leaching of plasticizers and their metabolites, phenolics, and pesticides from a range of human specimen collection and storage supplies. The objectives of this study were: 1) to develop methods to analyze environmental chemicals in supplies used for the collection and storage of human specimens, under simulated ‘normal’ and ‘worst-case’ scenarios, 2) to determine leaching rates and potential contamination levels, and 3) to evaluate the effect of storage duration and temperature on the leaching rates of target chemicals.

2. Materials and methods

2.1. Chemicals

A priori list of 121 environmental chemicals was targeted for analysis (Table S1). These chemicals were some of the commonly measured analytes in urine for the ECHO program, and were chosen for their relevance in human health. The 121 analytes include 45 plasticizers and their metabolites (phthalate metabolites and organophosphate triesters), 45 environmental phenols (bisphenols, benzophenones, parabens, chlorophenols and hydroxylated polycyclic aromatic hydrocarbons) and 31 current use pesticides (neonicotinoids, fungicides and herbicides) and their metabolites. Analytical standards for 121 target analytes along with 92 isotopically labeled internal standards were acquired from various suppliers (see Table S1). High-performance liquid chromatography (HPLC)-grade phosphoric acid was purchased from Burdick & Jackson (Muskegon, MI, USA). HPLC-mass spectrometer (MS) grade water and methanol were purchased from Fisher Scientific Co (Pittsburgh, PA, USA).

2.2. Samples

All collection and storage supplies were provided by Fisher Bioservices (Rockville, MD, USA), which were originally collected during 2019–2020. The supplies included 9 broad categories of products: 1) Cryovials (1 to 10 mL volume) used for low-temperature storage of biological samples (made of polypropylene, see Table S2). 2) Redtop serum tubes and K2EDTA tubes used for blood collection and processing (polypropylene). 3) Centrifuge tubes used for the separation of fluids or liquids (polypropylene). 4) Collection bottles and Samco specimen cups used for collection and storage of a variety of biological specimens (polypropylene). 5) Luer Lok sterile syringes and transfer pipettes (polyethylene). 6) Various gauges of needles (18G-23G) used for blood collection (contained metal and plastic parts). 7) Cotton pads, diapers, and toilet hats used in the collection of urine and stool, especially from infants. 8) Nitrile gloves. 9) Plastic bags such as biohazard specimen bags used for transport, storage, and disposal of biospecimens. A total of 1020 individual samples (i.e., devices) of these supplies were received in two batches from Fisher Bioservices during 2021 (N = 540) and 2022 (N= 480). Lot numbers were provided for each sample type (Table S2).

2.3. Sample/device extraction

Five replicates of each sample type from a single lot were analyzed using two extraction scenarios and these extractions were performed sequentially. The first scenario was extraction using a mixture of water and methanol (9:1, v/v, i.e., 10% methanol), which represented a typical leaching from devices of chemicals into biospecimens. The second scenario involved extraction with 100% methanol, a worst-case scenario for leaching. Each sample type was extracted sequentially in 10% methanol and 100% methanol and these two extracts were injected separately into LC-MS/MS.

For all sample types, a standardized extraction procedure was followed (see supporting information section S1.1 for details). In general, each sample was filled with an appropriate volume of solvent (10% methanol or 100% methanol), vortexed, and shaken in an orbital shaker for 1 h. The cryovials, serum tubes, large tubes and cups were then inverted and stored at room temperature for 24 h whereas syringes and transfer pipettes were left inverted for 10 min. For needles, a clean syringe was used to draw the extraction solvent slowly through the needle. The interior of the toilet hat was rinsed with the solvent five times, which was then analyzed. Post-leaching, a portion of the extract was aliquoted for LC-MS/MS analysis. Whereas a sequential extraction with 10% methanol followed by 100% methanol was used for most supplies, modifications were required for diapers, cotton pads, gloves, and plastic bags, for which a portion of samples was cut and used for two separate extractions.

2.4. Chemical analysis

Each extract was analyzed using two different LC-MS/MS methods as reported earlier (Zhu et al., 2021). The instrumental parameters used for the analysis are shown in Table S3. Method 1 was optimized for the detection and quantification of plasticizers, and some pesticides. Method 2 was used for the analysis of environmental phenols and remaining plasticizers and pesticides.

Instrumental analysis was performed on an HPLC system connected to an AB SCIEX QTRAP 5500+ triple quadrupole mass spectrometer, operating in both positive and negative ionization modes. For Method 1, target analytes were separated using an Ultra AQ C18^® column (2.1 mm × 100 mm, 3 μm) with mobile phases consisting of 0.1% acetic acid in water (A) and 0.1% acetic acid in methanol (B). The flow rate was 0.38 mL/min and the injection volume was 5.0 μL. The mobile phase flow was set at B 5% for 1 min followed by anincrease of B to 45% in over 0.2 min, held for 1.3 min and then ramped to 70% B in 2.2 min, and to 99% B in 2.0 min, and held for 3.0 min. The mobile phase was returned to initial conditions in 2.3 min for a total run time of 12 min. The MS/MS analysis was performed in negative ionization mode.

Method 2 entailed separation of target analytes on a Betasil^™ C18^® column (100 mm × 2.1 mm, 5 μm) with HPLC grade water (A) and methanol (B) as mobile phases. The flow rate was 0.38 mL/min and the injection volume was 5.0 μL. The following mobile phase gradient program was used: starting with 20% B for 1 min, ramped to 60% B in over 0.2 min, followed by an increase to 99% B in 4.3 min, held for 1.5 min then to 20% B in 0.5 min and equilibrated for 1.5 min, for a total run time of 9 min. The MS/MS analysis was conducted in both negative and positive ionization modes.

2.5. Quality assurance/quality control (QA/QC)

Procedural blanks were analyzed with every batch of samples to monitor for background levels of contamination arising from laboratory reagents and materials. Labware that contained any background levels of target analytes was either replaced or underwent additional cleaning. Isotopically labeled internal standards were added to each sample. A 12-point calibration curve with concentrations of target analytes ranging from 0.01 to 150 ng/mL, was generated for each batch of sample analysis. Quantification was by an isotope-dilution method. For those products such as vials, tubes, syringes and bottles which have specific surface contact areas with the extraction solvents, the results were quantitatively presented as the amount (as ng/sample) of each target analyte found. The surface area of the product varied depending on the size of the material. For cotton pads, gloves, diapers, and plastic bags (the contact surface area of these products with the solvent was difficult to establish), the weight of the product was determined, and the results were reported as ng/g of the product. Each lot number of the samples was analyzed in quintuplicate. Matrix spike tests were performed with known concentrations of target analytes fortified on select sample matrixes. The method detection limit (MDL) was determined as ten times the signal-to-noise ratio from the baseline of procedural blanks, and as the average signal in blanks plus three times the standard deviation when the target chemical was present in blanks.

2.6. Statistical analyses

The distribution of the data was examined by skewness and kurtosis values. The absolute values of skewness and kurtosis were generally greater than 1, indicating that the data distribution was not normal (Table S4). Given the non-normal distribution of the data, the Kruskal-Wallis test was used to assess the variability in chemical concentrations across different lot # of same sample type. The Mann-Whitney U test was used to compare the concentrations of target chemicals between two distinct sample types, and those received in 2021 and 2022.

2.7. Estimation of potential contamination concentration introduced from supplies

For vials, tubes, syringes, and needles, potential contamination concentrations (C_P1, ng/mL) introduced into biospecimens were calculated from the measured mass of target analytes leached from the supplies (C_M1, ng/sample) divided by the estimated volume of sample present in those containers (V_S1, e.g. urines and serum, m), using the equation (1):

$$C_{\text{P}1}(\text{ng}/\text{mL})=C_{\text{M}1}(\text{ng}/\text{sample})/V_{\text{S}1}(\text{mL}/\text{sample})$$ (1)

The volume of urine or serum was assumed to be at the full capacity of the container; for example, 3.0 mL sample for a vial with a capacity for 3-mL.

For gloves, diapers, and plastic bags, the estimated contamination concentration in samples (C_P2, ng/sample) was calculated from the concentration of target analytes measured in supplies (C_M2, ng/g), weight of diaper or plastic bag (W_S2, g/sample), and transfer rate (f, assumed to be 1% in this study) as shown in equation (2). This approach estimates the amount of chemicals potentially transferred from supplies to samples:

$$C_{\text{P}2}(\text{ng}/\text{sample})=C_{\text{M}2}(\text{ng}/\text{g})\times W_{\text{S}2}(\text{g}/\text{sample})\times f$$ (2)

The weight of diapers/plastic bags was measured using a precision scale.

For cotton pads, potential contamination per volume of urine collected (C_P3, ng/mL) was calculated based on the concentration measured in the product (C_M3, ng/g), weight of the product (W_S3, g/sample), and volume of urine collected (V_S3, mL) as shown in equation (3):

$$C_{\text{P}3}(\text{ng}/\text{mL})=C_{\text{M}3}(\text{ng}/\text{g})\times W_{\text{S}3}(\text{g}/\text{sample})/V_{\text{S}3}(\text{mL})$$ (3)

The weight of cotton pads was measured using a precision scale and the volume of urine in cotton pad was presumed to be 10 mL.

3. Results and discussion

3.1. Leaching of analytes in 10% methanol

Majority of the 121 target analytes were found either below the detection limit (BDL) or present at concentrations < 1.0 ng/sample in the leachates of 10% methanol (Table S5). The detection frequencies of analytes were generally below 10% for environmental phenols and pesticides. However, high detection frequencies of mono-ethyl phthalate (mEP) and triethyl phosphate (TEP) were found in cryovials. mEP is a biological metabolite of diethyl phthalate (DEP), a widely used plasticizer (especially in cellulose acetate based plastics) and a solvent in fragrances and cosmetics (Amin et al., 2018). Although mEP is primarily a biological metabolite of DEP, it can also be formed through photolysis of DEP (Peng et al., 2013). The 1-mL cryovials and cotton pads contained a mean mEP concentration of 0.06 ng/device and 41 ng/g, respectively. Although the occurrence of DEP in medical devices is reported earlier (Wang and Kannan 2023), no previous studies have measured mEP in specimen collection supplies. Notable concentration of mEP in cotton pads, that are commonly used for the collection of urine from infants/newborns, is concerning. Similarly, TEP was found at notable concentrations in cotton pads (GM: 8.0 ng/g), diapers (7.3 ng/g), nitrile gloves (77 ng/g), and 3 types of plastic bags (210–6300 ng/g), with a detection frequency of up to 100%. TEP is used as a plasticizer and a flame retardant in several plastic products. Mono-(2-ethylhexyl) phthalate (mEHP) and parabens are other contaminants detected in 10% methanol extracts of several collection supplies. In particular, cotton pads contained few ng/g levels of methyl-, ethyl- and propyl parabens (Table 1). Overall, cotton pads contained several target analytes at notable concentrations.

Table 1.

Geometric mean (GM) concentrations of select organic environmental chemicals leached from various human specimen collection and storage devices in 10% methanol (N = 1020).

Typea	N	mEPb	mIBP/mBP	mEHP	DNBP/DIBP	DPhP	TEP	TNBP/TIBP	TCEP	BADG-H2O	BADG-2H2O	MeP	EtP	PrP	BP-3	BPA	BPS
	ng/mL
1-mL cryovials	50
2-mL cryovials	45
2-mL cryovials, ext	75
3-mL cryovials	65	0.02					0.28							0.01
10-mL cryovials	55	0.01													0.01
18g needle	30	0.01				0.03
21g needle	95	0.02			0.03	0.02	0.01	0.03								0.03
22g needle	20	0.03		0.01						0.12	0.24					0.14
23g needle	10				0.02	0.04	0.03	0.02								0.03
centrifuge tube	25
collection bottle	25											0.06	0.18			0.02
cotton pad	15	5.6	1.3	0.36			1.7		1.5			3.1	1.9	2.1	0.26		0.35
fine tip transfer pipette	5	0.46	0.92	0.97
k2edta tube	95
syringe	25					0.01
redtop serum tube	85														0.01
smco Specimen Cup	80	0.01
toilet hat	20	0.01	0.03
transfer pipette	60	0.05		0.03		0.10
	ng/sample
biohazard bag	20	6.7				15	9400									12
biohazard specimen gag	5	0.97					44							0.24
leakproof bag	5	0.46					160							0.34			0.31
diaper	60	2.1			1.3	1.6	3.1							0.86			0.82
glove	50	0.59					11										0.10

Value below detection limits were leaved empty.

^aFor sample type details see Table S2.

^bFor chemical names see Table S1.

3.2. Leaching of analytes in 100% methanol

The extraction of sample collection supplies with 100% methanol showed elevated concentrations of several target analytes (Figure S1) such as TEP, tributyl phosphate (TNBP)/triisobutyl phosphate (TIBP), triphenyl phosphate (TPhP), tris(2-butoxyethyl) phosphate (TBOEP), BPA and bisphenol S (BPS), which exhibited median concentrations, in general, above 1 ng/device or 1 ng/g (Figure S2). The GM (mean ± SD) concentrations of TEP and TBOEP in diapers were 13 (66 ± 110) ng/g and 5.4 (7.7 ± 6.5) ng/g (Table S7), respectively, which were higher than those reported for food packaging (mean: 31 ng/g and 2.7 ng/g) (Zhou et al., 2023). BPA was detected at a GM concentration of 25 ng/g in biohazard plastic bags. Prevalence of these chemicals at such notable concentrations underscores the importance of prescreening of sample collection supplies prior to use in biomonitoring studies.

Approximately 30 of the 121 target analytes were detected in 100% methanol leachates of >50% samples (Table S7). Plastic bags, diapers, cotton pads, and gloves contained notable concentrations of plasticizers particularly OPEs and PAE metabolites. The variations in mEP and TEP concentrations across different types of samples are illustrated in Figure 1. The concentrations of TEP were notable among 3 types of plastic bags, with GM values ranging from 540 to 12,000 ng/g. These bags are often used for storage/package/envelop and transportation of samples.

Figure 1.

Amount and concentrations (mean ± SD) of mEP (a metabolite of diethyl phthalate) and TEP (organophosphate ester) in various sample collection and storage devices (N = 1020).

There were variations in concentrations of analytes among different sizes (i.e., capacity) of the same category of the product. For instance, 2-mL cryovials did not leach measurable levels of target analytes, but 1-mL and 3-mL vials contained notable concentrations of TEP. Furthermore, 1-mL cryovials contained notable concentrations of mEP and TNBP/TIBP. The 10-mL cryovials had detectable levels of mEP and methylparaben (MeP). Thus, there were varying levels of target analytes even among the same type of products of different sizes.

Certain syringes and transfer pipettes contained trace levels of BPA (Table S7). Parabens and bisphenols were detected in cotton pads and collection bottles. In collection bottles (for breast milk collection), ethyl paraben (EtP) was prevalent (GM: 95 ng/sample). Parabens are widely used as antimicrobial preservatives in consumer products (Zhu and Kannan 2020, Li et al., 2021) and their occurrence in collection bottles raises concern regarding contamination of biospecimens. Ethyl paraben was suggested as a preservative in urine storage containers ((Improving the Collection and Management of Human Samples Used for Measuring Environmental Chemicals and Nutrition Indicators (cdc.gov).

3.3. Variations among lot numbers

The variability in target analyte concentrations across various lot # of the same sample type was examined (Table S9). For some analytes, we found relatively consistent leaching across different lot # of the same sample type (for example, mMP and mEP in Samco specimen container). Conversely, significant differences in analyte concentrations among various lot # of the same sample type were found for several analytes (for example, OPEs and parabens in diapers). Similarly, analyte concentrations among several lot # of 1-mL cryovials and diapers varied widely (Figure S4). The lot-to-lot variation in analyte concentration could be related to raw materials and/or manufacturing processes used. These results suggest the need for screening individual lots to assess the suitability of supplies for sample collection and storage.

We also found that leaching patterns of some analytes varied depending on the lot # of supplies shipped in 2021 and 2022 (Table S10). mEP, TNBP/TIBP, EtP, and BPB were found at higher concentrations in 1-mL cryovials and diapers from 2021 than those from 2022 (Figure 2). TEP concentrations were higher in various lot # of 1-mL cryovials and diapers from 2022 than those from 2021. These temporal differences could be attributed to changes in manufacturing processes, raw materials, or other environmental factors. Conditions of the manufacturing processes for commercial devices and containers can vary with each batch produced.

Figure 2.

Concentrations of environmental chemicals leached from one-mL cryovials and diapers of different lot # in 100% methanol. Only data with significant differences (p < 0.05) are presented. For one-mL cryovials, Round 1 consisted of 40 samples from 8 lot numbers and Round 2 consisted of 10 samples from 2 lot numbers. For diapers, Round 1 consisted of 15 samples from 3 lot numbers and Round 2 consisted of 45 samples from 9 lot numbers.

3.4. Influence of storage time and heat/temperature on leaching of analytes

Factors such as storage temperature and duration can affect leaching of chemicals from plastic containers (Gulizia et al., 2023). Because 1-mL cryovials, contained notable concentrations of several target analytes, we examined this product further, by assessing the influence of storage duration and temperature on leaching of target analytes.

After the second extraction, half of the one-mL cryovials (n = 25) were filled with 0.5 mL of 100% methanol and stored for three months at room temperature. An aliquot (90 μL) of the extract was transferred on days 36 and 101 and fortified with internal standards for analysis. Overall, the concentrations of most target analytes did not vary over the period of 3 months (Figure S5). However, a significant increase (p < 0.05) in leaching of MeP, TEP, 4-hydroxybenzophenone (4-OH-BP) and TNBP/TIBP was found with an increase in storage duration. A study showed that migration of phthalate diesters from plastic pipes into water was influenced by temperature, pH, and duration (Zhang et al., 2023). Our finding suggests that prolonged storage of samples can exacerbate the leaching of certain chemicals from cryovials.

Similarly, one-mL cryovials (n = 25) were filled with 0.5 mL of 100% methanol and stored at 37.5 °C, and 75°C for 60 min. An aliquot (90 μL) of the extract was transferred into a vial for LC-MS/MS analysis. The concentrations of most target analytes remained relatively unchanged across different temperatures, but the concentrations of parabens, 4OHBP, TEP, and TNBP/TIBP increased at higher temperatures. One study reported that elevated temperatures increase the leaching of di-n-butyl phthalate from microplastics (Li and Tang, 2023). These observations suggest that temperature and duration of storage can alter leaching of chemicals from storage vials.

3.5. Estimation of contamination levels introduced from supplies into specimens

In cryovials and tubes, the GM concentrations of analytes leached into 10% methanol were below MDL or <1.0 ng/mL, which were often below the levels found in human populations (Tables 1 and S6). The results imply that the risks of potential contamination introduced by these collection/storage supplies are minimal. However, the estimated GM concentrations of TEP in 10% methanol of cotton pads and diapers were 1.7 ng/mL and 3.1 ng/mL, respectively. TEP leached from plastic bags and nitrile gloves was in the range of 11- 9400 ng/sample. Similarly, the concentrations of BPA, TPhP, DNBP/DIBP, and DPhP were also high in plastic packing bags, with GM values ranging from below detection limit to 100 ng/sample. These results suggest that some supplies such as cotton pads, diapers, plastic bags and nitrile gloves can be a source of contamination, unless adequate precautions are taken during sample collection and storage.

Leaching studies with 100% methanol showed that mMP (GM = 0.22 ng/mL), mEP (0.37 ng/mL), TEP (11 ng/mL), TNBP+TIBP (0.65 ng/mL), and MeP (0.18 ng/mL) can be present in 1-mL cryovials at measurable concentrations (Tables 2 and S8). The GM concentration of these metabolites in urine from the US population was 29 ng/mL for mEP (Zhao et al., 2023). On the other hand, the concentrations of individual OPE metabolites were typically less than 1.0 ng/mL in human urine (Guo et al., 2022). Thus, studies that measure TEP in urine should consider potential contamination arising from the products. It should be noted that these comparisons of chemical concentrations reported in previous studies with those found in supplies are only for contextual understanding and do not imply that previous research suffered from contamination. Overall, our results suggest that contamination of samples with TEP released from collection/storage devices should be carefully considered.

Table 2.

Geometric mean (GM) concentrations of select organic environmental chemicals leached from various human specimen collection and storage devices in 100% methanol (N = 1020).

Typea	mEPb	mIBP/mBP	mEHP	DNBP/DIBP	DPhP	TEP	TNBP/TIBP	TCEP	TPhP	TBOEP	BADG-2H2O	MeP	EtP	PrP	BP-3	BPA	BPS
	ng/mL
1-mL cryovials	0.37					11	0.65	0.07				0.18	0.10	0.13
2-mL cryovials	0.13		0.13				0.23	0.03					0.01
2-mL cryovials, ext
3-mL cryovials	0.04					2.81	0.11	0.02				0.05	0.01	0.03	1.6
10-mL cryovials
18g needle	0.02
21g needle	0.03		0.01	0.17			0.42				0.08					0.13
22g needle	0.04		0.02								0.51	0.02				0.35
23g needle	0.01			0.08	0.05	0.10	0.14		0.02		0.07			BDL0		0.10
centrifuge tube												0.04
collection bottle	0.04	0.04	0.02		0.20							0.08	1.2		0.01	0.04
cotton pad	14	2.9	0.67			3.6	5.6	2.9		4.5		6.8	13	7.2	0.46		1.2
fine tip transfer pipette		1.90	1.62									1.4				0.96
k2edta tube												0.06
syringe	0.01					0.01					0.02			0.01
redtop serum tube												0.06			0.02
smco Specimen Cup
toilet hat	0.02	0.05										0.02	0.01
transfer pipette	0.08		0.06			0.06				0.06
	ng/sample
biohazard bag	8.76			4.87	21.61	17000	12		89					0.76		38	1.4
biohazard specimen gag	1.67					110	3.3	0.40		3.43			0.32	1.1			0.34
leakproof bag	0.77					330	4.6	0.68		4.53		0.88	0.17	2.1			1.1
diaper	3.4			3.81		5.7	10	1.5	7.7	2.3		1.6	1.06	4.9			3.0
glove	0.99					82	53	0.58	11	1.0		0.45					1.6

BDL: Below detection limits.

^aFor sample type details see Table S2.

^bFor chemical names see Table S1.

Among needles tested, bisphenol A diglycidyl ether (BADGE)-2H₂O (GM: 0.51 ng/mL) and BPA (0.35 ng/mL) were found in 22G needles. Although needle was a metal piece, it is likely that packaging material could contribute to contamination. BADGE is widely used as an epoxy resin in several consumer products (Xue et al., 2022). The reported concentrations of BADGE in urine were in the ranges of 1.2–9.0 ng/mL from the United States and 0.77–4.1 ng/mL from China (Wang et al., 2012, Xue et al., 2022). EtP was found at notable concentrations in collection bottles, with concentrations of up to 1.2 ng/mL (equivalent to 95 ng/sample). Fine-tip transfer pipettes leached measurable levels of mEHP, MeP and BPA (0.96 to 1.6 ng/mL). The reported median concentrations of EtP in children’s urine ranged from 0.15 to 2.8 ng/mL worldwide (Wei et al., 2021). Nitrile gloves and plastic bags contained notable concentrations of OPEs, parabens, and BPs (ranging from below detection limits to 17400 ng/mL). Cotton pads can also be a source of potential contamination, particularly OPEs and parabens, with GM concentrations ranging from below detection limits to 13 ng/mL.

4. Conclusions and implications

We recommend that collection and storage devices used for the collection of biospecimens are screened for contaminants prior to use or at the minimum unused collection containers of the same model and lot # are archived and submitted to the laboratory for subsequent testing (as field blanks). Furthermore, manufacturer, model number, and lot numbers of collection devices should be maintained until the biospecimen analysis is completed. Our results suggest that leaching levels of most analytes from the tested supplies in 10% methanol were below the typical concentrations found in human biospecimens, except for cotton pads and storage bags. However, under the worst—case leaching scenario, several plasticizers and environmental phenols were found at notable concentrations. This study underscores the importance of prescreening of materials used in biospecimen collection, to ensure accuracy in trace level organic chemical analysis. Lot to lot variation in contamination levels of environmental chemicals in supplies further highlights the need for lot screening of these products. Furthermore, prolonged storage at high temperatures can exacerbate leaching of certain chemicals from collection materials.

Figure 3.

Effect of temperature [20 °C (room temperature), 37.5 °C, and 75 °C] on leaching of environmental chemicals from one-mL cryovials in 100% methanol. Concentrations were normalized to that measured at 20 °C for comparison.

Highlights.

• 24 types of human specimen collection devices were screened for environmental chemical contamination.

• Cryovials, diapers, cotton pads and plastic bags leached few plasticizers into solvents.

• DEP metabolite, mEP, and organophosphate ester, TEP, were found in several devices.

• Lot to lot variation in target chemical concentrations was found.

• Storage duration and temperature can affect leaching of chemicals from these products.