Validating the EPA Method 1633 for Aqueous Samples

Per- and polyfluoroalkyl substances (PFAS) are a class of synthetic compounds discovered in the 1940s commonly used for their surfactant properties.1-3

Image Credit: dba87/Shutterstock.com

Commercial household products, such as nonstick cookware and cleaning supplies, food packaging, water-resistant clothes, and industrial products like automotive lubricants and electronics, are among their many applications.3,4

Because of their broad usage in consumer items and natural persistence, PFAS have leached into the air, soil, and water, resulting in widespread exposure. Several research studies have identified the presence of PFAS in a wide range of matrices (e.g., wastewater, groundwater, drinking water, blood, serum, air, and soil).4-10

Growing concern about the presence and persistence of these compounds in the environment necessitates using commercially available instruments to execute current and evolving regulatory methods simply and reliably.

The United States Environmental Protection Agency has prioritized the development of sensitive, regular, and robust studies for detecting PFAS in diverse matrices, with a primary focus on drinking water. Method 537.1 focuses on the detection of 18 PFAS chemicals in drinking water.11

The EPA later developed a more thorough list, Method 533, which concentrates on additional short-chain PFAS and fluorotelomer alternatives in drinking water.12

In August 2021, the EPA released Draft Method 1633 for analyzing 40 PFAS chemicals in various matrices. As of August 2023, Draft Method 4 has been issued with the finalized multi-laboratory validated study for aqueous sample analysis.13

This article presents validation results for EPA Method 1633 for aqueous matrices utilizing the PerkinElmer LX-50™ UHPLC System coupled with the PerkinElmer QSight® 210 Triple Quadrupole Mass Spectrometry System.

Experimental

Materials and Reagents

Wellington Laboratories provided the primary PFAS standards, extracted internal standards (EIS), and non-extracted internal standards (NIS), as shown in Table 1.

VWR supplied the LC/MS grade water (reagent water) and methanol (MeOH). Formic acid was purchased from Sigma Aldrich. Fisher Scientific supplied the ammonium hydroxide.

Solid phase extraction was performed using a PromoChrom SPE-03 machine equipped with a MOD-004 sample loading device. Phenomenex provided Strata PFAS, polymeric weak anion exchange stacked with graphitized carbon black, and SPE cartridges (200 mg/50 mg, 6 mL).

Sigma Aldrich supplied the 250 mL high-density polyethylene (HDPE) bottles used to prepare and extract all blanks, spiked blanks, field samples, and QC samples.

The HPLC autosampler employed PerkinElmer low-volume 300 µL polyethylene (PE) vials with Sigma Aldrich polyethylene vial caps. Polypropylene vials and caps are necessary to prevent PFAS compounds from adsorbing onto glass vials and to eliminate PFAS materials often found in HPLC vial septa.

Table 1. Target analytes extracted internal standards and non-extracted internal standards. Source: PerkinElmer

Analyte Abbreviation
Perfluorobutanoic acid PFBA
Perfluoropentanoic acid PFPeA
Perfluorohexanoic acid PFHxA
Perfluoroheptanoic acid PFHpA
Perfluorooctanoic acid PFOA
Perfluorononanoic acid PFNA
Perfluorodecanoic acid PFDA
Perfluoroundecanoic acid PFUnA
Perfluorododecanoic acid PFDoA
Perfluorotridecanoic acid PFTrDA
Perfluorotetradecanoic acid PFTeDA
Perfluorobutanesulfonic acid PFBS
Perfluoropentansulfonic acid PFPeS
Perfluorohexanesulfonic acid PFHxS
Perfluoroheptanesulfonic acid PFHpS
Perfluorooctanesulfonic acid PFOS
Perfluorononanesulfonic acid PFNS
Perfluorodecanesulfonic acid PFDS
Perfluorododecanesulfonic acid PFDoS
1H, 1H, 2H, 2H-Perfluorohexane sulfonic acid 4:2FTS
1H, 1H, 2H, 2H-Perfluorooctane sulfonic acid 6:2FTS
1H, 1H, 2H, 2H-Perfluorodecane sulfonic acid 8:2FTS
Perfluorooctanesulfonamide PFOSA
N-methyl perfluorooctanesulfonamide NMeFOSA
N-ethyl perfluorooctanesulfonamide NEtFOSA
N-methyl perfluorooctanesulfonamidoacetic acid NMeFOSAA
N-ethyl perfluorooctanesulfonamidoacetic acid NEtFOSAA
N-methyl perfluorooctanesulfonamidoethanol NMeFOSE
N-ethyl perfluorooctanesulfonamidoethanol NEtFOSE
Hexafluoropropylene oxide dimer acid HFPO-DA
4,8-Dioxa-3H-perfluorononanoic acid ADONA
Perfluoro-3-methoxypropanoic acid PFMPA
Perfluoro-4-methoxybutanoic acid PFMBA
Nonafluoro-3,6-dioxaheptanoic acid NFDHA
9-Chlorohexadecafluoro-3-oxanonane-1-sulfonic acid 9Cl-PF3ONS
11-Chloroeicosafluoro-3-oxaundecane-1-sulfonic acid 11Cl-PF3OUdS
Perfluoro(2-ethoxyethane)sulfonic acid PFEESA
3-Perfluoropropyl propanoic acid 3:3FTCA
2H, 2H, 3H, 3H-Perfluorooctanoic acid 5:3FTCA
3-Perfluoroheptyl propanoic acid 7:3FTCA
Extracted Internal Standard Abbreviation
Perfluoro-n-[13C4]butanoic acid 13C4-PFBA
Perfluoro-n-{13C5}pentanoic acid 13C5-PFPeA
Perfluoro-n-[1,2,3,4,6-13C5]hexanoic acid 13C5-PFHxA
Perfluoro-n-[1,2,3,4-13C4]heptanoic acid 13C4-PFHpA
Perfluoro-n-[13C8]octanoic acid 13C8-PFOA
Perfluoro-n-[13C9]nonanoic acid 13C9-PFNA
Perfluoro-n-[1,2,3,4,5,6-13C6]decanoic acid 13C6-PFDA
Perfluoro-n-[1,2,3,4,5,6,7-13C7]undecanoic acid 13C7-PFUnA
Perfluoro-n-[1,2-13C2]dodecanoic acid 13C2-PFDoA
Perfluoro-n-[1,2-13C2]tetradecanoic acid 13C2-PFTeDA
Perfluoro-1-[2,3,4-13C3]butanesulfonic acid 13C3-PFBS
Perfluoro-1-[1,2,3-13C3]hexanesulfonic acid 13C3-PFHxS
Perluoeo-1-[13C8]octanesulfonic acid 13C8-PFOS
Perfluoro-1-[13C8]octanesulfoamide 13C8-PFOSA
N-methyl-d3-perfluoro-1-octanesulfonamidoacetic acid D3-NMeFOSAA
N-ethyl-d5-perfluoro-1-octanesulfonamidoacetic acid D5-NEtFOSAA
1H, 1H, 2H, 2H-Perfluoro-1-[1,2-13C3]hexanesulfonic acid 13C2-4:2FTS
1H, 1H, 2H, 2H-Perfluoro-1-[1,2-13C3]octanesulfonic acid 13C2-6:2FTS
1H, 1H, 2H, 2H-Perfluoro-1-[1,2-13C2]decanesulfonic acid 13C2-8:2FTS
Tetrafluoro-2-heptafluoropropoxy-13C3-propanoic acid 13C3-HFPO-DA
N-methyl-d7-perfluorooctanesulfonamidoethanol D7-NMeFOSE
N-ethyl-d9-perfluoroctanesulfonamidoethanol D9-NEtFOSE
N-ethyl-d5-perfluoro-1-octanesulfonamide D5-NEtFOSA
N-methyl-d3-perfluoro-1-octanesulfonamide D3-NMeFOSA
Nonextracted Internal Standard Abbreviation
Perfluoro-n-[2,3,4-13C3]butanoic acid 13C3-PFBA
Perfluoro-n-[1,2,3,4-13C4]octanoic acid 13C4-PFOA
Perfluoro-n-[1,2-13C2]decanoic acid 13C2-PFDA
Perfluoro-n-[1,2,3,4-13C4]octanesulfonic acid 13C4-PFOS
Perfluoro-n-[1,2,3,4,5-13C5]nonanoic acid 13C5-PFNA
Perfluoro-n-[1,2-13C2]hexanoic acid 13C2-PFHxA
Perfluoro-1-hexane[18O2]sulfonic acid 18O2-PFHxS

 

Hardware/Software

The analytes were chromatographically separated using a PerkinElmer LX-50 UHPLC System and then detected using a PerkinElmer QSight 210 Triple Quadrupole Mass Spectrometer with electrospray ionization (ESI).

The LX50 Autosampler was altered by substituting all polytetrafluoroethylene (PTFE) based tubing with polyether ether ketone (PEEK) tubing to minimize or remove any PFAS compound contamination caused by the PTFE tubing.

In addition, a PEEK needle was fitted to the autosampler. PerkinElmer Simplicity™ 3Q software was used for all instrument control, acquisition, and processing.

Method

LC Conditions and MS Parameters

Table 2 shows the LC method and MS source parameters. This approach used a pair of C18 columns.

A delay column (PerkinElmer Brownlee™ SPP C18 Column, 50 x 3.0 mm, 2.7 µm) was installed in-line between the LX50 pump and the autosampler to catch and delay potential PFAS interference arising from the LC pump and solvent reservoirs.

To separate PFAS from other interfering components, an analytical column (Brownlee SPP C18 Column, 75 x 4.6 mm, 2.7 µm) and a guard column (Brownlee SPP C18 Column, 5 mm x 4.6 mm, 2.7 µm) were also employed.

The LC gradient program was adjusted from the one specified in EPA Method 1633, as shown in Table 3. The LC program reduces the runtime by two minutes compared to EPA 1633. In addition, the same procedure may be utilized for examination of both 537.1 and 533.14,15

The MS source parameters, which include gas flows, temperature, and position settings, were tuned to achieve optimal sensitivity.

Table 4 shows how the compound-dependent parameters, such as collision energy (CE), entrance voltage (EV), and collision cell lens voltage (CCL2), were optimized for the target compounds. Wherever possible, a secondary mass transition was chosen for confirmation.

Table 2. LC Method and MS Source Conditions. Source: PerkinElmer

LC Conditions
Analytical Column Brownlee SPP C18 Column, 75 x 4.6 mm, 2.7 μm (PN: N9308415)
Guard Column Brownlee SPP C18 Column, 5mm x 4.6 mm, 2.7 μm (PN: N9308532)
Delay Column Brownlee SPP C18 Column, 50 x 3.0 mm, 2.7 μm (PN: N9308408)
Mobile Phase A 10 mM ammonium acetate in water
Mobile Phase B Methanol
Flow Rate 0.8 mL/min
Column Oven Temperature (°C) 40
Auto Sampler Temperature (°C) 15
Injection Volume 10
Needle Wash 1 25% acetonitrile in methanol
Needle Wash 2 50% water in methanol
MS Source Conditions
Electrospray Voltage -3000
Drying Gas 110
Nebulizer Gas 400
Source Temperature (°C) 200
HSID Temperature (°C) 280

 

Table 3. LC Gradient Program. Source: PerkinElmer

Step # Time (min) Mobile Phase A (%) Mobile Phase B (%)
1 0.00 95 5
2 0.70 95 5
3 1.00 55 45
4 7.00 2 98
5 8.00 2 98
6 8.10 95 5
7 10.00 95 5

 

Table 4. Optimized MRM Parameters for the PFAS analytes, extracted internal standards and non-extracted internal standards. Source: PerkinElmer

Acronym Ret Time
(min)
Precurser
Ion (m/z)
Product
Ion (m/z)
CEa EVb CCL2c Quantifier/
Qualifier
Type
13C4-PFBA 2.82 217.0 172.0 14 -4 40 Quantifier EIS
13C3-PFBA 2.82 216.0 172.0 14 -4 40 Quantifier NIS
PFBA 2.82 213.0 169.0 13 -9 36 Quantifier Analyte
PFMPA 3.06 229.0 85.0 30 -10 70 Quantifier Analyte
13C5-PFPeA 3.51 268.0 223.0 15 -12 45 Quantifier EIS
PFPeA 3.51 263.0 219.0 15 -8 80 Quantifier Analyte
3:3FTCA 3.56 241.0 184.0 30 -3 110 Quantifier Analyte
3:3FTCA 3.56 241.0 117.0 30 -3 110 Qualifier Analyte
13C3-PFBS 3.6 302.0 80.0 67 -28 80 Quantifier EIS
13C3PFBS 3.6 302.0 99.0 41 -28 80 Qualifier EIS
PFBS 3.6 299.0 80.0 65 -40 240 Quantifier Analyte
PFBS 3.6 299.0 99.0 40 -40 240 Qualifier Analyte
PFMBA 3.73 279.0 85.0 30 -10 55 Quantifier Analyte
PFMBA 3.73 279.0 97.0 40 -10 55 Qualifier Analyte
PFEESA 3.89 315.0 135.0 30 -30 75 Quantifier Analyte
PFEESA 3.89 351.0 83.0 30 -30 75 Qualifier Analyte
NFDHA 4.1 201.0 85.0 23 -5 30 Quantifier Analyte
13C2-4:2FTS 4.16 329.0 81.0 53 -36 60 Quantifier EIS
13C2-4:2FTS 4.16 329.0 309.0 29 -36 60 Qualifier EIS
4:2FTS 4.16 327.0 81.0 38 -4 65 Quantifier Analyte
4:2FTS 4.16 327.0 307.0 23 -1 110 Qualifier Analyte
13C2-PFHxA 4.22 315.0 270.0 15 -9 48 Quantifier NIS
13C5-PFHxA 4.22 318.0 273.0 12 -4 52 Quantifier EIS
PFHxA 4.22 313.0 269.0 17 -10 55 Quantifier Analyte
PFHxA 4.22 313.0 119.0 31 -10 50 Qualifier Analyte
PFPeS 4.26 349.0 80.0 73 -6 100 Quantifier Analyte
PFPeS 4.26 349.0 99.0 45 -6 90 Qualifier Analyte
13C3-HFPO-DA 4.42 287.0 169.0 12 -3 44 Quantifier EIS
13C3-HFPO-DA 4.42 287.0 185.0 29 -3 52 Qualifier EIS
HFPO-DA 4.42 285.0 169.0 10 -4 70 Quantifier Analyte
HFPO-DA 4.42 285.0 185.0 38 -4 70 Qualifier Analyte
13C4-PFHpA 4.84 367.0 322.0 17 -6 75 Quantifier EIS
PFHpA 4.84 363.0 319.0 16 -10 56 Quantifier Analyte
PFHpA 4.84 363.0 169.0 23 -10 92 Qualifier Analyte
13C3-PFHxS 4.86 402.0 80.0 87 -8 100 Quantifier EIS
13C3-PFHxS 4.86 402.0 99.0 40 -8 100 Qualifier EIS
18O2-PFHxS 4.86 403.0 103.0 38 -10 90 Quantifier NIS
PFHxS 4.86 399.0 80.0 85 -45 120 Quantifier Analyte
PFHxS 4.86 399.0 99.0 49 -45 87 Qualifier Analyte
ADONA 4.92 377.0 251.0 17 -10 62 Quantifier Analyte
ADONA 4.92 377.0 85.0 64 -10 62 Qualifier Analyte
5:3FTCA 4.99 341.0 237.0 24 -10 110 Quantifier Analyte
5:3FTCA 4.99 341.0 217.0 46 -10 110 Qualifier Analyte
13C2-6:2FTS 5.38 429.0 409.0 33 -16 124 Quantifier EIS
13C2-6:2FTS 5.38 429.0 81.0 43 -16 124 Qualifier EIS
6:2FTS 5.38 427.0 81.0 65 -8 80 Qualifier Analyte
6:2FTS 5.38 427.0 407.0 30 -8 30 Qualifier Analyte
PFHpS 5.38 449.0 80.0 86 -18 90 Qualifier Analyte
PFHpS 5.38 449.0 99.0 52 -18 80 Qualifier Analyte
13C4-PFOA 5.39 417.0 372.0 17 -5 80 Quantifier NIS
PFOA 5.39 413.0 369.0 20 -10 68 Quantifier Analyte
PFOA 5.39 413.0 169.0 30 -10 80 Qualifier Analyte
13C8-PFOA 5.39 421.0 376.0 16 -4 84 Quantifier EIS
13C4-PFOS 5.83 503.0 80.0 105 -45 125 Quantifier NIS
13C8-PFOS 5.84 507.0 80.0 107 -22 140 Quantifier EIS
13C8-PFOS 5.84 507.0 99.0 50 -22 140 Qualifier EIS
PFOS 5.84 499.0 80.0 100 -50 170 Quantifier Analyte
PFOS 5.84 499.0 99.0 55 -50 170 Qualifier Analyte
13C9-PFNA 5.85 472.0 427.0 20 -10 75 Quantifier EIS
13C5-PFNA 5.85 468.0 423.0 18 -14 70 Quantifier NIS
PFNA 5.85 463.0 419.0 20 -10 75 Quantifier Analyte
PFNA 5.85 463.0 219.0 29 -10 90 Qualifier Analyte
7:3FTCA 6.03 441.0 317.0 35 -10 190 Quantifier Analyte
7:3FTCA 6.03 441.0 337.0 24 -10 190 Qualifier Analyte
9Cl-PF3ONS 6.05 533.0 351.0 35 -30 110 Quantifier Analyte
9Cl-PFONS 6.05 533.0 83.0 35 -30 119 Qualifier Analyte
PFNS 6.23 549.0 80.0 80 -5 110 Quantifier Analyte
13C6-PFDA 6.25 519.0 474.0 17 0 88 Quantifier EIS
13C2-PFDA 6.25 515.0 470.0 16 -10 88 Quantifier NIS
PFDA 6.25 513.0 469.0 21 -10 90 Quantifier Analyte
PFDA 6.25 513.0 219.0 28 -10 96 Qualifier Analyte
13C2-8:2FTS 6.27 529.0 81.0 76 -40 115 Quantifier EIS
13C2-8:2FTS 6.27 529.0 509.0 35 -40 115 Qualifier EIS
8:2FTS 6.27 527.0 81.0 70 -3 100 Quantifier Analyte
8:2FTS 6.27 527.0 507.0 25 -30 90 Qualifier Analyte
d3-NMeFOSAA 6.43 573.0 419.0 27 -25 105 Quantifier EIS
NMeFOSAA 6.44 570.0 419.0 28 -20 100 Quantifier Analyte
NMeFOSAA 6.44 570.0 483.0 19 -20 100 Qualifier Analyte
PFDS 6.56 599.0 80.0 80 -3 130 Quantifier Analyte
13C7-PFUnA 6.58 570.0 525.0 21 -4 87 Quantifier EIS
PFUnA 6.59 563.0 519.0 19 -10 96 Quantifier Analyte
PFUnA 6.59 563.0 270.0 30 -10 85 Qualifier Analyte
d5-NEtFOSAA 6.6 589.0 419.0 28 -20 112 Quantifier EIS
d5-NetFOSAA 6.6 589.0 531.0 28 -20 105 Qualifier EIS
NEtFOSAA 6.6 584.0 419.0 30 -20 100 Quantifier Analyte
NetFOSAA 6.6 584.0 482.0 22 -20 99 Qualifier Analyte
13C8-PFOSA 6.7 506.0 78.0 35 -5 110 Quantifier EIS
PFOSA 6.7 498.0 78.0 48 -3 110 Quantifier Analyte
11Cl-PF3OUdS 6.71 631.0 451.0 37 -25 170 Quantifier Analyte
11Cl-PF3OUdS 6.71 631.0 199.0 32 -40 148 Qualifier Analyte
13C2-PFDoA 6.87 615.0 570.0 17 -14 104 Quantifier EIS
PFDoA 6.87 613.0 569.0 17 -10 104 Quantifier Analyte
PFDoA 6.87 613.0 319.0 30 -10 100 Qualifier Analyte
PFTrDA 7.12 663.0 619.0 18 -6 104 Quantifier Analyte
PFTrDA 7.12 663.0 369.0 40 -6 125 Qualifier Analyte
d7-NMeFOSE 7.31 623.0 59.0 37 -11 83 Quantifier EIS
d5-NEtFOSA 7.32 515.0 169.0 35 -3 110 Quantifier EIS
NMeFOSA 7.32 512.0 219.0 30 -3 120 Quantifier Analyte
NMeFOSA 7.32 512.0 169.0 30 -3 120 Qualifier Analyte
NMeFOSE 7.32 616.0 59.0 35 -1 105 Quantifier Analyte
13C2-PFTeDA 7.33 715.0 670.0 18 -7 140 Quantifier EIS
PFDoS 7.33 669.0 80.0 45 -5 110 Quantifier Analyte
PFDoS 7.33 669.0 119.0 45 -5 100 Qualifier Analyte
PFTeDA 7.33 713.0 669.0 19 -4 120 Quantifier Analyte
d9-NEtFOSE 7.5 639.0 59.0 40 -11 84 Quantifier EIS
NEtFOSE 7.51 630.0 59.0 19 -1 109 Quantifier Analyte
d3-NMeFOSA 7.52 531.0 169.0 38 -2 110 Quantifier EIS
NEtFOSA 7.52 526.0 219.0 30 -1 120 Quantifier Analyte
NEtFOSA 7.52 526.0 169.0 30 -1 120 Qualifier Analyte

a. CE = Collision Cell Energy
b. EV = Entrance Voltage
c. CCL2 = Collision Cell Lens 2 voltage

Calibration Standards Preparation

A mixed secondary stock of the target analytes was created from the five stock solutions, which was then used to generate ten calibration standards. Each calibration standard received an equal volume of Extracted Internal Standards (EIS) and Non-Extracted Internal Standards (NIS).

Based on the individual compound’s initial concentration in the stock solution, alyte concentration in calibration standards varied from 1 to 500,000 ng/L. For UHPLC analysis, calibration standards were moved to low-volume polypropylene vials with caps.

A wide range of calibration standards was employed to establish method linearity and instrument limits of detection (LOD). However, a smaller range and number of calibrants with a higher minimum level can be used in general practice. The EPA approach simply requires a minimum of six calibration standards.

Method Blanks, Fortified Samples, and Field Samples

All laboratory method blanks and fortified samples were created in 250 mL polypropylene bottles. A constant volume of EIS was spiked to each sample to assess extraction effectiveness based on recoveries.

A mixed analyte stock solution was utilized to spike known quantities of the target analytes into the fortified samples at different amounts to test and confirm analyte recoveries. This approach also determined the method detection limits (DL) and minimum level of quantitation (ML).

The SPE sample preparation procedure outlined in Section 12.0 of EPA Method 1633 extracted and concentrated all method blanks and fortified samples. Slight changes were made as permitted by the procedure.

The stacked GCB/WAX cartridge replaced the loose carbon step, reducing sample preparation time and the risk of analyte loss. The final 5 mL extracts were spiked with a consistent quantity of NIS before being transferred to vials with caps for analysis by LC/MS-MS.

Method blanks were routinely tested to guarantee the appropriate reduction or absence of PFAS interferences. Field samples were gathered from three non-potable sources in the Baltimore Metropolitan region. All field samples were collected in 250 mL polyethylene bottles and processed using SPE.

Solid Phase Extraction and Sample Concentration

PromoChrom SPE-003, an automated SPE system, was used. Extractions were carried out in line with the method defined in section 12.0 of EPA Method 1633. Stacked Weak Anion Exchange/Graphitized Carbon Black SPE 6 mL tubes containing 200 mg/50 mg sorbent, respectively.

The SPE approach followed the procedures shown in Figure 1. Because the method is performance-based, users elected to employ stacked GCB/ WAX cartridges instead of carbon cleanup, reducing both sample preparation time and the possibility of recovery losses.

Sample
Preparation
  • 250 mL sample
  • Add EIS directly
  • Check pH 6.0-7.0
Extraction
Setup
  • Silanized glass wool packed to half height of SPE cartridge
Condition
SPE
  • 15 mL of 1% methanolic ammonium hydroxide
  • 5 mL of 0.3M formic acid
Load
Sample
  • 5 mL/min
Rinse
  • 5 mL of reagent water (x2)
  • 5 mL 1:1 0.1M formic acid:MeOH
  • Dry under vacuum for 15 sec
Elute
  • Rinse sample bottle with 5 mL 1% NH4OH in MeOH
Filter
  • Install a nylon syringe filter on a 5 mL poly syringe
  • Decant sample supernatant into syringe barrel
Internal
Std
  • Add NIS to collection tube
Analysis
  • Transfer an aliquot into AS vial

 

Figure 1. Steps of Solid Phase Extraction Method. Image Credit: PerkinElmer

Results and Discussion

Remediation of PFAS Background Contamination

One of the most significant issues associated with PFAS trace analysis is contamination from chemicals, SPE apparatus, sample collection materials, volumetric ware, vials, the LC/MS/MS system, and the lab environment.

Many of these interferences can be attributed to the materials used in the manufacturing of volumetric ware, pipettes, syringes, tubing, and vials, as well as from PTFE components in the LC/MS/MS system.

To prevent or reduce these interferences from the LC/MS/MS system, a delay column was installed between the pump’s mobile phase mixer and the autosampler’s sample valve to trap and delay any PFAS chemicals released by the pump and mobile phase solvents.

This effectively separates the PFAS chromatographic peaks in the sample from the incoming PFAS contaminant peaks from the pump system. To prevent potential contamination, the LX50 autosampler's regular PTFE tubing was replaced with PEEK tubing.

All the materials used in this research work were checked for PFAS contamination before running tests by injecting blank samples. These experiments proved that all of the supplies utilized were free of PFAS contamination.

LC and MS/MS Methods

The QSight MS/MS MRM parameters were optimized for each analyte, EIS, and NIS using direct infusion experiments with a syringe pump.

Wherever possible, one precursor and product mass were identified, and the entrance voltage (EV), collision cell energy (CE), and collision cell lens 2 voltage (CCL2) were optimized for each compound in Simplicity 3Q. Table 4 shows the optimal MRM parameters.

Once the retention periods for each analyte were determined, a time-managed MRM MS/MS technique was utilized, with optimized time windows and dwell times to ensure at least ten scans across each analyte peak. The LC gradient method was adjusted to achieve good analyte separation, reduce run time, and improve peak symmetry.

A high-efficiency superficially porous particle (SPP) column was selected to give narrow peaks and short run times.

Figure 2 shows the total ion chromatogram (TIC). Figure 3 depicts the initial demonstration of the LC method capabilities, which created a baseline separation of branched vs. linear isomers for PFHxS, PFOS, PFOA, NMeFOSAA, and NEtFOSAA.

Total Ion Chromatogram of mid level calibration point containing (A) analytes, (B) non-extracted internal standards, and (C) extracted internal standards

Figure 2. Total Ion Chromatogram of mid level calibration point containing (A) analytes, (B) non-extracted internal standards, and (C) extracted internal standards. Image Credit: PerkinElmer

MRM chromatograms of PFHxS, PFOS, PFOA, NMeFOSAA, and NEtFOSAA showing the baseline separation of linear and branched chain isomers

Figure 3. MRM chromatograms of PFHxS, PFOS, PFOA, NMeFOSAA, and NEtFOSAA showing the baseline separation of linear and branched chain isomers. Image Credit: PerkinElmer

Table 5. Instrument and Method Calibration Ranges and Linearity (R2) for eight-point calibration curves of all EPA Method 1633 analytes. Source: PerkinElmer

Compound Instrument Calibration Range (ng/L)a R2b
PFBA 4-100,000 0.99988
PFMPA 2-50000 0.99992
PFPeA 2-50000 0.99997
3:3FTCA 3-70000 0.99720
PFBS 1-25000 0.99989
PFMBA 2-50000 0.99984
PFEESA 2-50000 0.99988
NFDHA 2-50000 0.99965
4:2FTS 4-100000 0.99953
PFHxA 1-25000 0.99983
PFPeS 1-25000 0.99989
HFPO-DA 2-50000 0.99909
PFHpA 1-25000 0.99980
PFHxS 1-25000 0.99951
ADONA 2-50000 0.99958
5:3FTCA 15-350000 0.99972
6:2FTS 4-100000 0.99900
PFHpS 1-25000 0.99968
PFOA 1-25000 0.99989
PFOS 1-25000 0.99984
PFOSA 1-25000 0.99915
PFNA 1-25000 0.99936
7:3FTS 15-350000 0.99949
9Cl-PF3ONS 2-50000 0.99990
PFNS 1-25000 0.99968
PFDA 1-25000 0.99947
8:2FTS 4-100000 0.99945
NMeFOSAA 1-25000 0.99969
PFDS 1-25000 0.99952
PFUnA 1-25000 0.99984
NEtFOSAA 1-25000 0.99941
11Cl-PF3OUdS 2-50000 0.99901
PFDoA 1-25000 0.99915
PFTrDA 1-25000 0.99925
NMeFOSA 1-25000 0.99966
NMeFOSE 10-250000 0.99984
PFDoS 1-25000 0.99914
PFTeDA 1-25000 0.99928
NEtFOSE 10-250000 0.99966
NEtFOSA 1-25000 0.99934

a. Instrument calibration range is the actual concentration range of calibration
standards used to determine calibration curves.
b. R2 values are the average of triplicate calibration curves.

Table 6. Instrument limits of detection (LOD) and limits of quantitation (LOQ) for all target analytes in EPA Method 1633. Source: PerkinElmer

Analyte Instrument (ng/L)a
LOD LOQ
PFBA 10 30
PFMPA 1 4
PFPeA 5 15
3:3FTCA 50 160
PFBS 1 4
PFMBA 3 9
PFEESA 2 5
NFDHA 1 4
4:2FTS 1 4
PFHxA 4 12
PFPeS 1 4
HFPO-DA 20 60
PFHpA 0.5 2
PFHxS 1 4
ADONA 0.8 3
5:3FTCA 0.3 1
6:2FTS 4.2 14
PFHpS 1 3
PFOA 2 5
PFOS 0.5 2
PFNA 6 20
7:3FTCA 5 17
9Cl-PF3ONS 10 34
NMeFOSAA 6 21
PFNS 2 6
PFDA 20 65
8:2FTS 4 13
NEtFOSAA 4 12
PFDS 1 4
PFUnA 10 45
11Cl-PF3OUdS 4 14
PFOSA 0.5 2
PFDoA 20 67
PFTrDA 9 29
PFDoS 30 87
PFTeDA 4 14
NMeFOSA 7 24
NMeFOSE 20 53
NEtFOSE 5 16
NEtFOSA 8 25

a. Instrument LOD/LOQ was determined using the signal-to-noise ratio (S/N) of the peak from the lowest detectable calibration standard and extrapolating to the concentration at which the S/N = 3 or 10 for LOD or LOQ, respectively. This is an estimate to demonstrate expected LOD/LOQ and can vary from lab to lab.

Linearity, Instrument Limits of Quantitation (LOQ), and Instrument Limits of Detection (LOD)

Calibration curves were utilized to determine linearity, instrument limits of detection (LOD), and limits of quantitation (LOQ) for all PFAS targets and surrogates.

Three replicates were used to create ten-point calibration curves in the concentration range of ~1 – 500,000 ng/L, using non-weighted linear regression with the intercept set to zero.

Table 5 shows that all analytes had correlation coefficient values (R2) of more than 0.99, indicating excellent linearity over the studied range of concentrations.

The instrument limits of detection (LOD) and quantitation (LOQ) for each target analyte were established at the lowest detectable standard on the calibration curve (ng/L), which was then extrapolated to yield a signal-to-noise ratio (S/N) of 3 for LOD and 10 for LOQ.

Table 6 summarizes the instrument and method limits of detection (LODs) and limits of quantification (LOQs).

Triplicate injection calibration curves for representative analytes PFBA, PFOA, PFOS, NMeFOSA.

Figure 4. Triplicate injection calibration curves for representative analytes PFBA, PFOA, PFOS, NMeFOSA. Image Credit: PerkinElmer

Determination of Method DLs, MRLs

The method detection limits (DL) and minimum quantitation (ML) levels were calculated using EPA Method 1633. Replicate reagent water samples were spiked at three varying levels and used in computations. Each fortified sample received a constant volume of EIS.

Each of these fortified samples was processed using the full sample preparation approach. Aliquots of each sample were then moved to polypropylene vials and examined using an LC/MS/MS system to measure analyte and EIS recoveries.

Table 7 shows that the recoveries for all analytes at all fortification levels ranged between 94 and 106%. The recovery RSDs were less than 10% across all fortification levels. All recoveries were within the limits specified by the EPA method for the extracted internal standards.

The method DLs and MLs were computed and validated using the statistical analysis approaches outlined in EPA Method 1633. Table 8 presents the determinations of DLs and MLs.

Table 7. PFAS analyte recovery data for fortified of reagent water spiked at two different levels. Five replicate samples were extracted at each fortification level. Source: PerkinElmer

  Low Fortification Level (n=5) High Fortification Level (n=5)
Fortified
Conc.
(ng/L)
%
Average
Recovery
%RSD Fortified
Conc.
(ng/L)
%
Average
Recovery
%RSD
PFBA 16 100 4 64 106 2
PFMPA 8 106 2 32 99 6
PFPeA 8 97 3 32 99 5
3:3FTCA 16 103 6 64 99 5
PFBS 4 101 7 16 98 5
PFMBA 8 94 5 32 99 3
PFEESA 8 96 4 32 98 5
NFDHA 8 99 6 32 101 3
4:2FTS 16 96 2 64 94 3
PFHxA 4 99 6 16 97 4
PFPeS 4 99 5 16 98 4
HFPO-DA 8 97 3 32 98 4
PFHxS 4 98 4 16 99 2
PFHpA 4 101 3 16 103 3
ADONA 8 101 3 32 100 2
5:3FTCA 80 98 4 320 95 2
6:2FTS 16 99 3 64 98 4
PFOA 4 101 2 16 100 2
PFHpS 4 103 5 16 103 2
PFOS 4 98 4 16 96 5
PFNA 4 102 1 16 101 3
7:3FTCA 80 104 2 320 104 3
9Cl-PF3ONs 8 98 4 32 99 4
NMeFOSAA 4 97 3 16 95 3
PFNS 4 98 4 16 101 3
PFDA 4 100 4 16 99 3
8:2FTS 16 101 3 64 99 4
NEtFOSAA 4 99 2 16 98 4
PFDS 4 100 4 16 101 2
PFUnA 4 95 3 16 102 3
11Cl-PF3OUdS 8 99 3 32 98 2
PFOSA 4 95 6 16 99 3
PFDoA 4 99 5 16 100 5
PFTrDA 4 102 6 16 99 4
PFDoS 4 97 5 16 100 5
PFTeDA 4 99 5 16 97 4
NMeFOSA 4 99 4 16 100 2
NMeFOSE 40 98 3 160 96 4
NEtFOSE 40 99 5 160 98 4
NEtFOSA 4 100 5 16 100 3

 

Table 8. Method detection limits (MDL) and minimum level of quantitation (ML) on the QSight 210 LC-MS/MS system. Experimental values are compared to reference values reported in EPA Method 1633. Source: PerkinElmer

Analyte QSight 210 EPA Method 1633c
MDLa (ng/L) MLb (ng/L) MDL (ng/L) ML (ng/L)
PFBA 1.33 5.0 0.79 2.0
PFMPA 0.43 2.0 1.46 5.0
PFPeA 1.21 5.0 0.54 2.0
3:3FTCA 3.04 10.0 2.47 10.0
PFBS 1.30 5.0 0.37 2.0
PFMBA 1.16 5.0 1.41 5.0
PFEESA 0.35 2.0 1.17 5.0
NFDHA 0.41 2.0 0.75 2.0
4:2FTS 1.16 5.0 1.68 5.0
PFHxA 0.96 5.0 0.46 2.0
PFPeS 0.63 2.0 0.5 2.0
HFPO-DA 0.89 5.0 0.51 2.0
PFHxS 0.56 2.0 0.54 2.0
PFHpA 0.58 2.0 0.37 2.0
ADONA 0.71 2.0 0.50 2.0
5:3FTCA 0.25 2.0 9.59 20.0
6:2FTS 1.24 5.0 2.45 10.0
PFOA 1.10 5.0 0.54 2.0
PFHpS 1.12 5.0 0.50 2.0
PFOS 0.71 2.0 0.63 2.0
PFNA 1.04 2.0 0.45 2.0
7:3FTCA 1.04 2.0 8.71 20.0
9Cl-PF3ONS 3.51 10.0 1.38 5.0
NMeFOSAA 0.31 2.0 0.68 2.0
PFNS 0.30 2.0 0.47 2.0
PFDA 2.94 10.0 0.52 2.0
8:2FTS 0.74 2.0 2.5 10.0
NEtFOSAA 0.77 2.0 0.59 2.0
PFDS 0.61 2.0 0.60 2.0
PFUnA 0.56 2.0 0.45 2.0
11Cl-PF3OUdS 0.69 2.0 1.67 5.0
PFOSA 0.79 5.0 0.32 2.0
PFDoA 1.24 5.0 0.40 2.0
PFTrDA 0.51 2.0 0.46 2.0
PFDoS 2.86 10.0 0.6 2.0
PFTeDA 0.65 2.0 0.49 2.0
NMeFOSA 0.51 2.0 0.43 2.0
NMeFOSE 0.46 2.0 3.81 10.0
NEtFOSE 0.62 2.0 4.84 20.0
NEtFOSA 0.48 2.0 0.45 2.0

a. Experimental DL was determined from seven LFB replicates fortified at ~4-16 ng/L measured over three days and calculated according to procedure 40 CFR Part 136, Appendix B.
b. Experimental MLs were calculated by multiplying the MDL by 3.18 and rounding to the nearest, 1, 2 or 5 x 10n as specified in section 9.2.2 of 1633.
c. Values taken directly from Table 8 of 1633 Draft 4.

Field Sample Analysis

Field samples were gathered from three distinct locations in the Baltimore Metropolitan region and labeled S1, S2, and S3. Three samples were taken from each location. Before extraction, all samples were spiked with a consistent amount.

All samples collected at a single location were extracted, evaporated, and reconstituted using SPE technology. The reconstituted samples were subsequently spiked with NIS, and an aliquot was moved to a polypropylene vial for LC/MS/MS testing.

Table 9 summarizes the results from all samples. At least one PFAS compound was identified in each sample. The first sample contained PFOS, while the second sample had PFBA, PFBS, PFHpA, and PFOS. PFBA and PFHpA were detected in sample 3.

All other analytes were either undetectable or below the MLs, as indicated by <ML in the table.

Conclusion

This article describes the validation of an LC/MS/MS technique for determining PFAS analytes and isotopically labeled standards listed in the US EPA Method 1633. This technique uses the PerkinElmer LX50 UHPLC System in conjunction with the PerkinElmer QSight 210 Triple Quadrupole Mass Spectrometer.

All PFAS analytes and isotopic standards demonstrated excellent linearity, with R2 values of ≥ 0.99. The instrument LODs and LOQs confirm that the QSight 210 LC/MS/MS System possesses the necessary sensitivity to quantify the PFAS analytes.

To eliminate and minimize background PFAS pollutants, instrument modifications and the insertion of a delay column are required, and the effectiveness of these modifications has been verified using blank analysis.

An enhanced chromatographic technique previously developed for EPA Method 537.1 to reduce LC/MS/MS runtimes has been demonstrated to work for 533 analysis and is now being used for 1633 analysis.14,15

The QSight 210 LC/MS/MS System was used to optimize MRM experiments for all analytes and isotopically labeled standards, including quantifier and qualifier MRMs where feasible.

A time-managed MRM mass spectrometer approach has also been optimized to enhance dwell time for increased sensitivity while retaining more than ten data points per chromatographic peak.

Recoveries for all analytes range between 94 and 106%, while recoveries for extracted internal standards all fall within the values defined by method 1633. The SPE extraction in the current study was performed using an automated SPE system. Non-potable water samples from three separate locations were collected and examined.

Overall, this validation study demonstrates that the LX50 UHPLC system combined with the QSight 210 tandem quadrupole mass spectrometer is an effective analytical instrument for the application of EPA Method 1633, with sufficient sensitivity to assess all analytes.

In addition, a singular method, which used the same columns and mobile phases, was validated for analyzing all analytes from EPA Methods 1633, 533, and 537.1 without physically altering the system or consumables other than using a different SPE cartridge and preparation technique as specified by each methodology.

This will significantly enhance throughput in labs that need to analyze drinking water using either EPA method.

Table 9. Average concentration of PFAS from each sampling location. Source: PerkinElmer

Analyte Average Concentration
S1 S2 S3
PFBA <ML 12±2 13±1
PFMPA <ML <ML <ML
PFPeA <ML <ML <ML
3:3FTCA <ML <ML <ML
PFBS <ML 1.5±0.5 <ML
PFMBA <ML <ML <ML
PFEESA <ML <ML <ML
NFDHA <ML <ML <ML
4:2FTS <ML <ML <ML
PFHxA <ML <ML <ML
PFPeS <ML <ML <ML
HFPO-DA <ML <ML <ML
PFHxS <ML <ML <ML
PFHpA <ML 0.7±0.1 0.62±0.02
ADONA <ML <ML <ML
5:3FTCA <ML <ML <ML
6:2FTS <ML <ML <ML
PFOA <ML <ML <ML
PFHpS <ML <ML <ML
PFOS 2.0±0.3 8±1 <ML
7:3FTCA <ML <ML <ML
9Cl-PF3ONS <ML <ML <ML
NMeFOSAA <ML <ML <ML
PFNS <ML <ML <ML
PFDA <ML <ML <ML
8:2FTS <ML <ML <ML
NEtFOSAA <ML <ML <ML
PFDS <ML <ML <ML
PFUnA <ML <ML <ML
11Cl-PF3OUdS <ML <ML <ML
PFOSA <ML <ML <ML
PFDoA <ML <ML <ML
PFTrDA <ML <ML <ML
PFDoS <ML <ML <ML
PFTeDA <ML <ML <ML
NMeFOSA <ML <ML <ML
NMeFOSE <ML <ML <ML
NEtFOSE <ML <ML <ML
NEtFOSA <ML <ML <ML

 

References and Further Reading

  1. Kissa, E., Fluorinated Surfactants and Repellents. Second Edition ed. Surfactant Science Series, ed. A. Hubbard. 2001, New York, NY: Marcel Dekker.
  2. Lau, Christopher, et. al. “Perfluoroalkyl Acids: A Review of Monitoring and Toxicological Findings.” Toxicological Sciences, 99, 2, 2007, pp 366-394. http://doi.org/10.1093/toxsci/kfm128
  3. https://www.epa.gov/pfas/basic-information-pfas
  4. Wang, Z. et al. “An Overview of the Uses of PFAS.” https://doi.org/10.1039/D0EM00291G
  5. Boiteux, V.; Bach, C.; Sagres, V.; Hemard, J.; Colin, A.; Rosin, C.; Munoz, J.-F.; Dauchy, X. Analysis of 29 Per- and Polyfluorinated Compounds in Water, Sediment, Soil and Sludge by Liquid Chromatography–Tandem Mass Spectrometry. Int. J. Environ. Anal. Chem. 2016, 96 (8), pp 705–728. https://www.tandfonline.com/doi/full/10.1080/03067319.2016.1196683
  6. Banzhaf, S.; Filipovic, M.; Lewis, J.; Sparrenbom, C.J.; Barthel, R. “A Review of Contamination of Surface-, Ground-, and Drinking Water in Sweden by Perfluoroalkyl and Polyfluoroalkyl Substances (PFASs).” Ambio. Springer Netherlands April 1, 2017, pp 335-346. https://doi.org/10.1007/s13280-016-0848-8
  7. Buck, R. C.; Franklin, J.; Berger, U.; Conder, J.M.; Cousnis, I.T.; Voogt, P. De’ Jensen, A.A.; Kaanan, K.; Mabury, S.A.; van Leeuwen, S.P.J. “Perfluoroalkyl and Polyfluoroalkyl Substances in the Environment: Terminology, Classification, and Origins.” Integr. Environ. Assess. Manag. 2011, 7(4), pp 513-541. https://setac.onlinelibrary.wiley.com/doi/10.1002/ieam.258#:~:text=(2006)%2C%20the%20presence%20of,of%20larger%20functional%20derivatives%20and
  8. Parry, E.; Anumol, T. “Quantitative Analysis of PFAS in Drinking Water Using Liquid Chromatography Tandem Mass Spectrometry.” The Column 2019, 15 (5), pp 9–15.
  9. Glaser, J.A.; Foerst, D.L.; McKee, G.D.; Quave, S.A.; Budde, W.L. “Trace Analyses for Wastewaters.” Environ. Sci. Technol. 1981, 15, pp 1426-1435.
  10. Maya E. Morales-McDevitt, Jitka Becanova, Arlene Blum, Thomas A. Bruton, Simon Vojta, Melissa Woodward, and Rainer Lohmann. Environmental Science & Technology Letters 2021 8 (10), 897-902. DOI: 10.1021/acs.estlett.1c00481
  11. Shoemaker, J.A.; Tettenhorst, D.R. “EPA Method 537.1: Determination of Selected Per- and Polyflourinated Alkyl Substances in Drinking Water by Solid Phase Extraction and Liquid Chromatography/Tandem Mass Spectrometry (LC/ MS/MS) Version 2.0” EPA Document #: EPA/600/R-20/006 March 2020.
  12. Rosenblum, L., Wendelken, S.C. “EPA Method 533: Determination of Per- and Polyfluoroalkyl Substances in Drinking Water by Isotope Dilution Anion Exchange Solid Phase Extraction and Liquid Chromatography/Tandem Mass Spectrometry” EPA Document #: 815-B-19-020 December 2019.
  13. Draft Method 4 1633 Analysis of Per- and Polyfluoroalkyl Substances (PFAS) in Aqueous, Solid, and Biosolids, and Tissue Samples by LC-MS/MS” EPA Document #: EPA 821-D-23-001 July 2023.
  14. Weisenseel, J., Costanzo, M. “Analysis of Perfluoroalkyl and Polyfluoroalkyl Substances in Drinking Water: Validation Studies of EPA Method 537.1 Using the QSight 220 UHPLC/ MS/MS” Application Note.
  15. Belunis, A., LaCourse, W., Costanzo, M. “Improved Throughput for the Analysis of Perfluoroalkyl and Polyfluoroalkyl Substances in Drinking Water by EPA Method 533” Application Note.

This information has been sourced, reviewed and adapted from materials provided by PerkinElmer.

For more information on this source, please visit PerkinElmer.

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