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 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 1x10-6 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)

 

GC-MS Conditions

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