1. Introduction
Over the last 20 years, active pharmaceutical ingredients (APIs) and their metabolites have been identified in all environmental compartments and their occurrence has raised concerns over potential impacts on ecosystem and human health [
1,
2,
3]. However, despite two decades of research, significant knowledge gaps exist regarding the environmental exposures to APIs [
1,
4]. For example, a complete or significant lack of knowledge exists for many parts of the world (e.g., Africa and South America) as such research disproportionally targets wealthy regions including North America, Western Europe and China [
4,
5]. In many poorly studied parts of the world, APIs are openly available without a prescription and prone to miss-/over-use so concentrations might be expected to be greater than those reported so far [
6]. Similarly, API disposal practices and inefficient wastewater connectivity and treatment may further exacerbate high API concentrations in some regions [
1,
6]. Underpinning such research are the complex analytical methods employed for specific quantification of APIs in water, namely High-Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MS/MS). Such instrumentation is required for obtaining environmentally relevant sensitivity in measurements of APIs, down to ng/L levels.
The high cost of necessary instruments and analytical intensity of the methods employed are key barriers to broadening measurement of APIs in the environment to a global scale. Furthermore, such barriers may magnify existing regional disproportionalities in published data. Of the available published data, among the most significant challenges for compiling an international perspective on APIs in the aquatic environment is the cross-comparison between datasets obtained via different methodologies. Key areas of deviation between methodologies include sample collection protocols (e.g., collection from the river bank vs. centroid flow), analytical techniques and statistical/quantitative interpretations and use of quality-control samples throughout collection and analysis. No single, unified analytical method exists, and in-house method validations are not always required for publication, making accurate and reliable interpretations of existing data on concentrations of APIs in different regions of the globe challenging.
Recent advances in the sensitivity of analytical instrumentation provides the ability to now analyse a wide range of APIs with minimal sample pre-treatment (e.g., Furlong et al. [
7]; Campos-Mañas et al. [
8]. Direct-injection HPLC-MS/MS is characterised by large-volume sample injections (100–5000 μL) which eliminate the need for sample pre-concentration [
9], traditionally achieved using solid phase extraction (SPE). This technique has been successfully used since the 1980s [
9] and more recently employed for quantification of pharmaceuticals, illicit drugs and other organic contaminants in surface and wastewater [
7,
8,
10]. Such methods significantly reduce the volume of sample needed (simplifying both sample collection and shipment to the laboratory) and the time for sample analysis via elimination of complex pre-treatment. Additionally, such methods offer a more environmentally responsible alternative to traditional methods (e.g., SPE) due to reduced sample volume and elimination of any need for solvents in sample pre-treatment. Direct-injection protocols also provide an opportunity to perform much larger-scale monitoring programmes than previously possible (e.g., due to financial and time constraints), allowing a better understanding of environmental exposures to pharmaceutical compounds (APIs and corresponding API metabolites) to aquatic systems around the globe. Conducting such large-scale global monitoring programmes will likely raise logistical challenges in terms of sample transport from the site of collection to the site of analysis. For these studies to succeed, therefore, it is important that sample integrity is maintained during such transport.
Here we present and evaluate a monitoring approach for use in large-scale monitoring programmes for APIs in multiple environmental matrices (e.g., WWTP influents, effluents, surface water and drinking water). The approach presents a simple and standardized set of protocols for the consistent collection, shipment, and analysis of aqueous samples using a uniform collection kit and a single HPLC-MS/MS analytical method. An interlaboratory evaluation of the protocol was conducted with surface water (i.e., river water) and WWTP effluent collected by the U.S. Geological Survey (USGS) from an effluent-impacted stream (Muddy Creek, North Liberty, Iowa, USA). This protocol may significantly reduce the challenges of: (a) evaluating spatial and temporal API concentration trends across variations in geography, climate, land use, hydrogeology, and demographics, (b) the lack of accessibility to costly analytical equipment and operating costs necessary for accurate and sensitive API quantification (namely HPLC-MS/MS) in some regions of the world, and (c) obtaining water samples from under-studied regions of the world for accurate, sensitive, reliable sample analysis. The development of this protocol therefore provides an opportunity to begin to better understand the risks of pharmaceuticals and other compounds at the global scale, a research priority highlighted in recent horizon scanning exercises on pharmaceuticals and chemicals more generally [
5,
11].
2. Materials and Methods
2.1. Test Substances
The protocol was developed for quantifying concentrations of 61 strategically selected APIs (
Table 1) representing 19 therapeutic classes of medicinal chemicals approved for use in humans (
n = 57) and animal husbandry (
n = 4). The study APIs were selected to include: (a) compounds of high usage across the world; (b) compounds with known or suspected ecological or human health concern; and (c) compounds of expected high use due to regional disease pressures (e.g., antimalarials). Significant focus was placed on antimicrobial chemicals including antibiotics (
n = 13) and antifungals (
n = 6) due to implications for the selection of resistance to these medicines in bacterial communities [
12,
13]. Similarly, focus was also placed on antidepressants (
n = 7) due to their increasing use globally and potential ecotoxicological risk [
14]. All compounds were optimised for specific quantification using direct injection HPLC-MS/MS only and compounds not suitable for quantification using this instrumentation were not included in the study. Further method development may be used to broaden the scope of the studied contaminants (e.g., Campos-Mañas et al. [
8]).
All test standards were purchased from Sigma Aldrich (UK) and were of ≥95% purity. Deuterated internal standards were obtained from Sigma Aldrich (UK) for 32 test APIs and atrazine-D5 was used where a labelled standard was not available. Liquid chromatography-mass spectrometry (LCMS)-grade water and methanol were obtained from VWR (UK). Polystyrene boxes (5 L, 34.5 × 21 × 14.5 cm, L × W × H) used for sample shipment were obtained from JB Packaging (Torpoint, UK), whereas 15-mL amber glass sample vials, 0.7-μm glass microfiber syringe filters (Whatman) and 24-mL luer lock syringes were obtained from Fisher Scientific (UK). A ZORBAX Eclipse Plus C18 chromatography column (3.0 × 100 mm, 1.8 μm, 600 bar) was purchased from Agilent Technologies (UK) and a C18 SecurityGuard guard column was purchased from Phenomenex (UK).
2.2. Sampling Kits and Water Collection Protocol
The sampling kits were designed to simplify logistics so that a large number of locations could be sampled with a minimum of effort. As the sample injection volume for the developed method was only 100 μL, the standard collection volume was set at 10 mL of sample water. Samples were collected in duplicate to provide a backup sample in case of breakage of the primary sample container during shipment. Each sampling kit therefore contained: 20 amber glass vials (15 mL) (for collection of 10 samples in duplicate), two ice packs, 10 polypropylene syringes (24 mL), 10 glass microfiber filters (0.7-μm pore size), a 500-mL stainless-steel bucket with 10-m long nylon cord attached, material to collect a field blank quality-control (QC) sample with LCMS-grade water, a standardised collection log and sample labels. Kits weighed 2.25 kg and were able to fit into a 34.5 × 21 × 14.5 cm box (approximately the size of a shoe box).
The sample collection protocol, including storage and shipping procedures, was included with the sampling kit and instructional videos were also provided online (<
https://youtu.be/HeZ7xoxJXhM> and <
https://youtu.be/PLyCNcVCKdc>). At each location, the bucket (included with each kit) was rinsed three times with native sample water prior to collection. Following sample collection, 20 mL of sample water was aspirated into the syringe, and the syringe filter was then attached and primed with 5 mL of sample water. Then, 5 mL of filtrate was used to rinse out a 15-mL vial prior to dispensing the remaining 10 mL of filtrate into the glass vial. This procedure was repeated once more with the same bucket of water to create the second replicate. At this point, pH, temperature or other probes may be inserted into the water remaining in the bucket to obtain additional environmental data. All vials were labelled with their location, sample date/time, and replicate number and immediately placed on ice upon collection. Prior to shipment, samples were frozen until being shipped on ice to the University of York (York, UK) Centre of Excellence in Mass Spectrometry (CoEMS) using DHL global express delivery (1–2 days). Freezing the samples prior to express shipping to CoEMS ensured the samples maintained their integrity (i.e., did not become warm during shipment) prior to analysis.
2.3. HPLC-MS/MS Protocol
The analytical method was adapted from a previously developed method for pharmaceuticals compounds [
7]. Analysis occurred by direct-injection (100-µL injection volume) HPLC-MS/MS in multiple reaction monitoring (MRM) mode with positive electrospray ionisation. A Thermo Scientific Endura TSQ triple quadrupole mass spectrometer coupled with a Thermo Scientific Dionex UltiMate 3000 HPLC was used for all analyses. Two transition ions were optimised (for collision energy and retention time) in-house, one for quantitation (T1) and another for confirmation (T2) of precursor identity (
Table S1). The instrument-calibrated fragmentor voltage was used for all analysis. Mobile phase A was LCMS-grade water with 0.01 M formic acid and 0.01 M ammonium formate while mobile phase B was 100% methanol. The flow rate was 0.45 mL/min. Flow was diverted away from the spectrometer for the first 1 min of the analytical run to avoid poorly retained materials (e.g., slats) from reaching the nebuliser. The HPLC gradient started at 10% B which increased to 40% at 5 min, 60% at 10 min, and 100% at 15 min, where it remained until 23 min then reduced to 10% at 23.1 min prior to a 10-min re-equilibration time between runs. Autosampler temperature was maintained at 6 °C while the column temperature was maintained at 40 °C. The collision gas was argon and was set at a pressure of 2 mTorr. Quantification occurred using a 15-point calibration prepared for 33 deuterated internal standards (
Table 1,
Table S1) ranging from 1 to 8000 ng/L (
Table S2). All calibrants were made using a standard method as described by Furlong et al. [
7] in such a way as to maintain an equal proportion of methanol in the final calibrants (
Table S2). Atrazine-D5 was used where a labelled standard was not available for a specific target chemical, as established by Furlong et al. [
7].
2.4. Quality Control
Extensive quality-control measures were used in-house and in the field to ensure that the laboratory and field protocols were not causing false positives or negatives in the corresponding environmental results. Materials needed to conduct one field blank were included in each sample kit which included 25 mL of LCMS-grade water, a syringe filter, syringe and two 15-mL glass vials. The procedure for collection, storage and shipment of this QC sample was exactly the same as for environmental samples, except using LCMS-grade water. This step enables an evaluation of sample contamination derived from collection in the field.
In addition to field QC measures, a blank as well as method and instrumental QCs were injected after every 10 injections during analytical runs. The laboratory blanks were pure LCMS-grade water with all internal standards spiked to a concentration of 400 ng/L. Both method and instrumental QCs consisted of all target APIs spiked in LCMS-grade water at a concentration of 80 ng/L with all QCs at 400 ng/L. However, the method QC underwent the same sample storage and preparation measures as actual samples and the instrumental QC was spiked directly into a HPLC vial prior to analysis. Before each use, the nebuliser and spray guard of the mass spectrometer were cleaned with methanol. Additionally, prior to an analytical run, the chromatography column was equilibrated with 20 injections of a composite environmental sample (made from equal aliquots of the samples being analysed in respective runs) in order to condition the chromatography column prior to analysis.
2.5. Method Validation
The method was validated based on USGS method No. O-2440-14 (National Water Quality Laboratory [NWQL] laboratory schedule 2440) for filtered water [
7], which will be referred to as USGS method No. 5-B10 in this paper. Briefly, intra-/inter-day repeatability was determined at three concentrations (10, 100 and 1000 ng/L) over 3 days (
n = 10 per concentration). Analyte response (recovery) was also determined at three concentrations (10, 100 and 1000 ng/L) in LCMS-grade water, drinking water directly from the tap (chlorinated), surface water, and WWTP influent and effluent. Surface water was obtained from the River Ouse in York City Centre (UK, GPS coordinates: 53.957397, −1.083816), drinking water was from the tap at the University of York (York, UK), and both WWTP influent and effluent were obtained from a WWTP in Barnsley, UK. Limits of detection (LOD) and quantification (LOQ) were statistically derived using the method described by Sallach et al. [
15]. Briefly, respective LODs were based on the Grubbs
t-test constant for 10 variables multiplied by the standard deviation of 10 replicate quantifications of test chemicals in mixture at the lowest calibrant level (1 ng/L). The LOQs for respective analytes were determined as two times the LOD [
15]. Analytical limits were determined in LCMS-grade water, drinking water, surface water, and WWTP influent and effluent. An acceptable range for analyte response was considered between 60–130% and <20% for intra-/inter-day repeatability and precision as established by USGS method No. 5-B10 [
7].
2.5.1. Evaluation of Chemical Stability
To evaluate the potential degradation of test APIs during shipment, a stability assessment was performed at three temperatures: 4 °C (
n = 6), 20 °C (
n = 6), and 35 °C (
n = 6) (
Table S3). Six replicates of 10-mL LCMS-grade water for each temperature were spiked with a mixture of all test chemicals to make a final mixed concentration of all the APIs at 1000 ng/L. Samples were then stored at the designated temperatures for either 2 or 7 days to provide the range of holding times from the field to the laboratory that would be encountered for this protocol. A 1-mL aliquot of each sample was collected, and analysis occurred via the same procedure as environmental samples. In addition to the stability assessment to assess if sample storage temperature during shipping affects API concentrations, the interior temperature of three sets of polystyrene packages containing two ice packs frozen at −20 °C was measured over 7 days to determine the conditions samples are likely to experience during shipment.
2.5.2. Interlaboratory Assessment
To validate the method using independent analytical results, an interlaboratory comparison was conducted where four samples (three stream samples and 1 WWTP effluent sample) were simultaneously collected for API analysis at CoEMS and the USGS using USGS method No. 5-B10 [
7]. Both methods used the exact same field sample processing procedures and materials (e.g., 15-mL amber glass sample vials, 24-mL leur lock syringes, and 0.7-µm glass microfiber syringe filters) making for a more effective comparison of the analytical methods used without having added variability due to field collection and processing procedures. All samples for this interlaboratory comparison (
Figure S1) were collected from Muddy Creek, North Liberty, Iowa (USA) using the provided sampling kit and designated protocols and were immediately chilled and express mailed the same day as sample collection and arriving within 24 h to the USGS NWQL in Denver (Colorado, USA) for analysis. Samples to CoEMS were frozen following collection and then express mailed where they were received within 37 h still frozen. There were 30 overlapping APIs between these two methods for this interlaboratory comparison (
Table S4). USGS method No. 5-B10 uses the same chromatography column, injection volume and positive ESI as the CoEMS method, however it is conducted using an Agilent Technologies 6460 triple quadrupole tandem mass spectrometer coupled to an Agilent 1200 HPLC system [
7]. The data were evaluated based on their absolute difference (%) and the order (from highest concentration to lowest) of quantified APIs.
