Combine EPA 521 and EPA 522 into a Single Analysis Using a (Very) Large Volume Injection with an Agilent Style Split/Splitless Inlet

While I was at NEMC in San Antonio, I posted a teaser for my drinking water talk here on the blog. I thought I would follow-up with some details for those of you who were unable to attend (and those who did). Today, I’m going to give my final run conditions for the 50 µL injections, which enabled me to detect nitrosamines in fortified laboratory samples at concentrations as low as 1.0 ng/L (with calibration low points equivalent to 0.2 ng/L). Periodically, I will make followup posts to explain some of the choices I made.

To evaluate the viability of a CSR-LVSI approach to meeting the IRIS 1×10-6 cancer risk levels, a calibration curve was prepared using 1,4-dioxane (cat.# 30287), Nitrosamine Calibration Mix, Method 521 (cat.# 31898), 8270 Appendix IX mix #1, revised (cat# 32459), THF (cat.# 30414). The surrogates 1,4-dioxane-d8 (cat.# 30614) and N-nitrosodimethylamine-d6 (cat.# 33910) and internal standards THF-d8 (cat.# 30112) and N-nitrosodipropylamine-d14 (cat.# 33911) were added at constant concentrations to each level in the calibration curve (Table I).

Table I – Calibration Curve (ng/mL)

ICAL 1

ICAL 2

ICAL 3

ICAL 4

ICAL 5

ICAL 6

ICAL 7

ICAL 8

THF-d8

100

100

100

100

100

100

100

100

THF

0.10

0.20

0.50

1.0

2.5

5.0

25

50

1,4-Dioxane-d8

200

200

200

200

200

200

200

200

1,4-dioxane

0.10

0.20

0.50

1.0

2.5

5.0

25

50

n-nitrosodimethylamine-d6

20

20

20

20

20

20

20

20

n-nitrosodimethylamine

0.005

0.01

0.025

0.050

0.125

0.25

1.25

2.5

n-nitrosomethylethylamine

0.010

0.020

0.050

0.10

0.25

0.50

2.5

5.0

n-nitrosodiethylamine

0.010

0.020

0.050

0.10

0.25

0.50

2.5

5.0

n-nitrosodi-n-propylamine-d14

10

10

10

10

10

10

10

10

n-nitrosopyrrolidine

0.010

0.020

0.050

0.10

0.25

0.50

2.5

5.0

n-nitrosodi-n-propylamine

0.010

0.020

0.050

0.10

0.25

0.50

2.5

5.0

n-nitrosomorpholine

0.005

0.010

0.025

0.050

0.125

0.25

1.25

2.5

n-nitrosopiperidine

0.010

0.020

0.050

0.10

0.25

0.50

2.5

5.0

n-nitrosodi-n-butylamine

0.010

0.020

0.050

0.10

0.25

0.50

2.5

5.0

Solid Phase Extraction (SPE)

To evaluate method performance across a variety of concentrations, three fortified samples were prepared at three different concentrations and used to evaluate recoveries. The deuterated N-nitrosodimethylamine surrogate was added at 400 ng/L so that the extracts would have a final surrogate concentration of 20 ng/mL. The deuterated 1,4-dioxane surrogate was added at 4000 ng/L with an expected final extract concentration of  200 ng/mL. The target reporting limits for 1,4-dioxane and THF are ten times higher than most of the nitrosamines, so calibration standards, matrix spike levels, and surrogate levels all reflect this difference. The bottled waters were fortified while still in their plastic bottles, recapped, mixed by inversion and allowed to sit for several hours to ensure homogeneous samples. See Table II for compound specific spike levels.

Each sample was extracted using a Resprep® activated coconut charcoal SPE cartridge following the procedure for SPE Option 1 – Extraction of 500-mL Samples described in section 11.4 of EPA Method 522. Immediately after solvent elution, the extracts were spiked with 50 µL of internal standard mix and brought up to 10 mL final volume resulting in a concentration of 10 ng/mL or 100 ng/mL in the extracts. The extracts were then transferred to a large storage vial and dried with anhydrous sodium sulfate.

Table II – Laboratory Fortified Blank (LFB) concentrations (ng/L) – Extract concentrations in parenthesis (ng/mL)

Blank Low Mid High
THF 0.0 (0.0) 10 (0.50) 50 (2.5) 500 (25)
1,4-Dioxane-d8 4000 (200) 4000 (200) 4000 (200) 4000 (200)
1,4-dioxane 0.0 (0.0) 10 (0.50) 50 (2.5) 500 (25)
n-nitrosodimethylamine-d6 0.40 (20) 400 (20) 400 (20) 400 (20)
n-nitrosodimethylamine 0.0 (0.0) 0.50 (0.025) 2.5 (0.13) 25 (1.3)
n-nitrosomethylethylamine 0.0 (0.0) 1.0 (0.050) 5.0 (0.25) 50 (2.5)
n-nitrosodiethylamine 0.0 (0.0) 1.0 (0.050) 5.0 (0.25) 50 (2.5)
n-nitrosopyrrolidine 0.0 (0.0) 1.0 (0.050) 5.0 (0.25) 50 (2.5)
n-nitrosodi-n-propylamine 0.0 (0.0) 1.0 (0.050) 5.0 (0.25) 50 (2.5)
n-nitrosomorpholine 0.0 (0.0) 0.50 (0.025) 2.5 (0.13) 25 (1.3)
n-nitrosopiperidine 0.0 (0.0) 1.0 (0.050) 5.0 (0.25) 50 (2.5)
n-nitrosodi-n-butylamine 0.0 (0.0) 1.0 (0.050) 5.0 (0.25) 50 (2.5)

 

GC-MS Conditions

CSR-LVSI image

Figure 1 – CSR-LVSI experimental setup

1. Single taper Restek Premium Liner with extra wool at the bottom, 2. 10m 0.53 mm ID Rxi deactivated guard, 3. SGE 0.4 – 0.8 mm OD µ-union, 4. 30m 0.25 mm ID x 1.0 µm df Rxi-5Sil MS, 5. 5975 MSD.

Table III– Agilent 7890A-5975C GC-MS parameters

7693 ALS Parameters
Syringe SGE 100 µL gas tight syringe with fixed 26/23 gauge needle – part number 005668
Injection Volume 50 µL
Injection Speed 4,000 µL/min
Split/Splitless GC Inlet Parameters
Inlet Mode Splitless for 1.5 min, then split 100 mL/min
Temperature 275 °C
Liner Custom Restek Premium 4mm ID Single Taper Liner with extra wool
GC Parameters
Flow Program 5.08 mL/min (hold 8.9 min) to 2.0 mL/min at 1.0 mL/min
Oven Temperature Program 35 °C (hold 1.5 min) to 50 °C at 50 °C/min (hold 7.1 min) to 320 °C at 11.12 °C/min (hold 1.5 min)
Pre-Column 0.53 mm ID x 10 m Rxi guard column – cat#10072
Analytical Column 30m x 0.25 mm ID x 1.0 µm df Rxi-5sil ms – cat#13653
Column Union SGE µ-Union 0.8 to 0.4, Part Number 073562
Carrier Gas He, constant flow
MS Parameters
Transfer Line Temp. 320 °C
Source Temp. 230 °C
Quad Temp. 150 °C
Electron Energy 70 eV
Solvent Delay Time 9.90 min
Tune Type BFB
Ionization Mode EI
SIM Program

Group

Start Time (min)

Ions (m/z)

Dwell (ms)

1

9.90

42, 46, 71, 72, 78, 80

20

2

10.50

58, 62, 64, 88, 96

20

3

11.20

42, 43, 46, 48, 74, 80

20

4

12.00

43, 56 88, 102

30

5

15.50

58, 68, 70, 78, 86, 100, 116, 130, 144

20

6

17.40

84, 99, 114, 116, 141, 158

20

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4 Responses to “Combine EPA 521 and EPA 522 into a Single Analysis Using a (Very) Large Volume Injection with an Agilent Style Split/Splitless Inlet”

  1. David Salvat says:

    I understand the role of the pre-column in making this large volume injection, but what about the large vapor cloud, from the injected solution, flashing outside of the inlet liner and possibly backflushing into the tubing connected to the inlet? Thanks.

  2. Some of the solvent vapor is surely lost to the split vent and septum purge; the analytes, however, are trapped in the wool by the cooling caused by the rapidly evaporating solvent. Theoretically, a large vapor cloud should never develop. Aside from the initial surge in pressure when the solvent starts evaporating, there should be a self-regulating process of solvent evaporation in the hot inlet and concurrent recondensation of solvent occurring on the cool pre-column (hence the name, Concurrent Solvent Recondensation – Large Volume Splitless Injection)

  3. In Table II a LFB (take NDMA, for example) with a concentration of 0.5 ug/L creates an extract at 0.025 ug/mL (when 500mL is extracted to 10mL), Table I shows ICAL 3 at 0.025ug/mL and ICAL 1 at 0.005ug/mL. ICAL 1 equates to a water sample at 0.1 ug/L. This is equal to a low calibrator at 100ng/L. How are detections at 1.0ug/L and 0.2ng/L (first paragraph in paper) confirmed? Are there some unit typos? Thanks.

  4. Some of the units were incorrect. The units should be ng/L and ng/mL (for extracts and calibration points). I’ve made the corrections in the text. The full application note can be found at http://www.restek.com/pdfs/EVAN1922A-UNV.pdf

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